Colon and Pancreas Cancer Peptidomimetics

ABSTRACT

The invention relates to a peptidomimetic of an NPC-1 epitope on the MUC5AC protein which is differentially expressed in pancreatic and colorectal cancer, and diagnostic and therapeutic usages. Further, antibodies that selectively bind the NPC-1 epitope peptidomimetics and may be used in diagnostic and therapeutic methods.

RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/633,184, filed Jun. 26, 2017, which is a Divisional of U.S. patentapplication Ser. No. 14/725,477, filed May 29, 2015, now U.S. Pat. No.9,718,866, which is a Divisional of U.S. patent application Ser. No.13/825,717 filed Mar. 22, 2013, now U.S. Pat. No. 9,068,014, which is aNational Stage Application of International Patent Appl. No.PCT/US2011/053064, filed Sep. 23, 2011, which claims priority to U.S.Provisional Appl. No. 61/467,896, filed Mar. 25, 2011, and to U.S.Provisional Appl. No. 61/435,163, filed Jan. 21, 2011, and to U.S.Provisional Appl. No. 61/435,176, filed Jan. 21, 2011, and to U.S.Provisional Appl. No. 61/407,112, filed Oct. 27, 2010, and to U.S.Provisional Appl. No. 61/385,587, filed Sep. 23, 2010, and is acontinuation-in-part of International Patent Appl. No. PCT/US2011/041502, each of which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING DISCLOSURE

This application includes as part of its disclosure a biologicalsequence listing which is being concurrently submitted through EFS-Web.Said biological sequence listing is contained in a file named“I143282o001605.txt” which was created on Nov. 25, 2019, and has a sizeof 120.398, bytes, and is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION Molecular Biology of Cancer

Cancer is caused by a malfunction in the growth control systems of acell. Cells control their growth via combination of proliferationinhibition by tumor suppressor genes (e.g., Retinoblastoma protein(pRb), p53) and proliferation activation by oncogenes (proto-oncogenes)(e.g., RAS, WNT, MYC, EKR, and TRK). A mutation in either a tumorsuppressor gene and/or a protooncogene in a cell results in unusuallyhigh rates of cell proliferation (e.g., a tumor cell). See Knudson(1971) Proc. Natl. Acad. Sci. USA 68(4): 820-823. The cell may exhibitearly signs of aberrant growth such as aberrant morphology or unusuallylarge size (hyperplasia). The tumor cells also may proliferate at ahigher than usual but not lethal rate, forming a growth, known as benigntumor (dysplasia). In later stages of cancer, the tumor cellsproliferate at an unusually high rate resulting in uncontrolled growththat threatens the health of the patient known as malignant tumors (orin situ cancer). Many tumors can “metastasize” or spread throughout thebody forming tumors. Metastasis is generally a sign of late stage,terminal cancer. Weinberg (September 1996) “How Cancer Arises”Scientific American 62-70.

Prostate cancer, lung cancer, and colorectal cancer are the three mostcommon cancers among men. Lung cancer, prostate cancer, liver cancer,and colorectal cancer are the leading causes of cancer deaths among men.Breast cancer, lung cancer, and colorectal cancer are the three mostcommon cancers among women. Lung cancer, breast cancer, and colorectalcancer are the leading causes of cancer death among women. CDCFeatures—United States Cancer Statistics (USCS) (2011). At present,there is an urgent need for diagnoses and therapies for colorectal,pancreatic, prostate, lung, liver, and breast cancer. For example, eachyear in the United States alone, more than 43,000 people are diagnosedwith pancreas cancer. National Cancer Institute (2010) “What You Need toKnow about Cancer of the Pancreas.” Although advancements in cancerdetection and therapy have been made over the last two decades, thecurrent options for early detection and treatment of cancer are limitedand there exists a great need for new methods and materials that providefor the detection and treatment of cancer, especially colorectal andpancreatic cancer.

MUC5AC

Mucins are high molecular weight glycoproteins with O-linkedoligosaccharides attached to serine or threonine residues of theapomucin protein backbone expressed in a cell and tissue-specificpattern in normal tissues. The mucin family includes proteins thatcontain tandem repeat structures with a high proportion of prolines,threonines, and serines (which constitute the PTS domain). Mucins arefurther defined by extensive glycosylation of the PTS domain throughGalNAc O-linkages at the threonine and serine residues. Each mucin has acentral region with a variable number of tandem repeat with about eightamino acid residues, but there is a little similarity. There are twostructurally and functionally distinct classes of mucins: secretedgel-forming mucins and transmembrane mucins. Secreted gel-forming mucinsinclude the products of the MUC2, MUC5AC, MUC5B and MUC6 genes. SeeKocer, et al. (2006) BMC Gastroenterology 6: 4; See also Hollingsworth &Swanson (2004) Nature Reviews 4: 45-60.

The human mucin (MUC) family consists of members designated MUC1 toMUC21-subclassified into secreted and transmembrane forms. The secretedmucins (e.g., MUC2, MUC5AC, MUC5B and MUC6) form a physical barrier,which acts as a mucous gel that provides protection for epithelial cellsthat line the respiratory and gastrointestinal tracts and form theductal surfaces of organs such as the liver, breast, pancreas, andkidney. The transmembrane mucins (e.g., MUC1, MUC4, MUC13 and MUC16)have a single membrane-spanning region and contribute to the protectivemucous gel through their ectodomains of O-glycosylated tandem repeatsthat form rod-like structures. Kufe (2009) Nature Reviews 9: 874-885.MUC5AC expression is found on apical epithelial cells of the mucusglands of gastric antrum and body, tracheobronchial epithelium,superficial epithelium of the gallbladder and endocervix epithelium.

MUC5AC is highly expressed in adenoma. See Kocer, et al. (2006) BMCGastroenterology 6: 4. Additionally, MUC5AC is expressed in tumors ofgastrointestinal, pancreatiobiloary, and endocervical origin (e.g.,colon, esophagus, liver, lung, pancreas, stomach, and uterus). See Lau,et al. (2004) Am. J. Clin Pathol. 122: 61-69. MUC5AC is also highlyexpressed in breast and gastric cancers. Zhang, et al. (1998) ClinicalCancer Research 4: 2669-2676. Further, MUC5AC glycan variants have beenassociated with pancreatic neoplasms. Haab, et al. (May 2010) Annals ofSurgery 251(5): 937-945. MUC5AC is aberrantly expressed by colorectalpolyps and colorectal carcinoma. Kocer, et al. (2006) BMCGastroenterology 6(4): 1-9. Thus, there exists a need in the art forepitope peptidomimetics thereof for use in diagnostic and therapeuticcompositions and methods for treating pancreatic and colorectal cancer.

SUMMARY OF THE INVENTION

The present invention provides peptidomimetics of a NPC-1 epitopederived from MUC5AC, including compositions comprising the same as wellas methods of manufacture and use.

In one embodiment, the invention provides an isolated polypeptidecomprising a polypeptide at least about 80% identical to the amino acidsequence of SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ is for L; X² isE or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K(SEQ ID NO: 2), wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is Tor I and X⁵ is Q or Y; SLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3), wherein X¹ isE or D; X² is W or Y; and X³ is T or I; FPEDYFRYTNQK (SEQ ID NO: 4);SLPDDWFRYINY (SEQ ID NO: 5); or any one of the amino acid sequences ofSEQ ID NOs: 6-24. In another embodiment, the invention provides apolypeptide that is at least about 90% identical to the amino acidsequence of SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ is for L; X² isE or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K(SEQ ID NO: 2), wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is Tor I and X⁵ is Q or Y; SLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3), wherein X¹ isE or D; X² is W or Y; and X³ is T or I; FPEDYFRYTNQK (SEQ ID NO: 4);SLPDDWFRYINY (SEQ ID NO: 5); or any one of the amino acid sequences ofSEQ ID NOs: 6-24. In another embodiment, the invention provides apolypeptide comprises the amino acid sequence of SX¹PX²DX³FRYX⁴NX⁵ (SEQID NO: 1), wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is T or Iand X⁵ is Q or Y; SX¹PX²DX³FRYX¹NX⁵K (SEQ ID NO: 2), wherein X¹ is forL; X² is E or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y;SLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3), wherein X¹ is E or D; X² is W or Y;and X³ is T or I; FPEDYFRYTNQK (SEQ ID NO: 4); SLPDDWPRYINY (SEQ ID NO:5); or any one of the amino acid sequences of SEQ ID NOs: 6-24. In afurther embodiment, the polypeptide may comprise the amino acid sequenceof FPEDYFRYTNQK (SEQ ID NO: 4). In a further embodiment, the polypeptidemay comprise the amino acid sequence of SLPDDWFRYINY (SEQ ID NO: 5)

In one embodiment, the invention provides an isolated fusion proteincomprising a polypeptide comprises the amino acid sequence ofSX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ is for L; X² is E or D; X³is Y or W; X⁴ is T or I and X⁵ is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO:2), wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is T or I and X⁵is Q or Y; SLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3), wherein X¹ is E or D; X²is W or Y; and X³ is T or I; FPEDYFRYTNQK (SEQ ID NO: 4); SLPDDWFRYINY(SEQ ID NO: 5); or any one of the amino acid sequences of SEQ ID NOs:6-24. In a further embodiment, the fusion protein may comprise apolypeptide comprising the amino acid sequence of FPBDYFRYTNQK (SEQ IDNO: 4). In a further embodiment, the the fusion protein may comprise apolypeptide comprising the amino acid sequence of SLPDDWFRYINY (SEQ IDNO: 5). In another embodiment, the isolated fusion protein may comprisea polypeptide that is at least about 80% or 90% identical to the aminoacid sequence of the amino acid sequence of SEQ ID NOs: 1-24. In anotherembodiment, the fusion protein may comprise a detectable labelcovalently or non-covalently directly or indirectly attached thereto. Inanother embodiment, the detectable label may be selected from the groupconsisting of polyHis tag, FLAG tag, MBP, GST protein, and GFP.

The invention also provides a conjugate comprising a polypeptidecomprises the amino acid sequence of SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1),wherein X¹ is for L; X² is B or D; X³ is Y or W; X⁴ is T or I and X⁵ isQ or Y; SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO: 2), wherein X¹ is for L; X² is Eor D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y; SLEPEX¹DWX²FRYX³NY(SEQ ID NO: 3), wherein X¹ is E or D; X² is W or Y; and X³ is T or I;FPEDYFRYTNQK (SEQ ID NO: 4); SLPDDWFRYINY (SEQ ID NO: 5); or any one ofthe amino acid sequences of SEQ ID NOs: 6-24, directly or indirectly,conjugated to a cytotoxic agent, a therapeutic agent, label,carbohydrate, carrier, immunoglobulin or immunoglobulin fragment, or animmunomodulatory agent. In another embodiment, the conjugate maycomprise a polypeptide with at least about 80% or 90% identical to theamino acid sequence of SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ isfor L; X² is E or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y;SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO: 2), wherein X¹ is for L; X² is E or D; X³is Y or W; X⁴ is T or I and X⁵ is Q or Y; SLBPEX¹DWX²FRYX³NY (SEQ ID NO:3), wherein X¹ is E or D; X² is W or Y; and X³ is T or I; FPEDYFRYTNQK(SEQ ID NO: 4); SLPDDWFRYINY (SEQ ID NO: 5); or any one of the aminoacid sequences of SEQ ID NOs: 6-24, directly or indirectly, conjugatedto a cytotoxic agent, a therapeutic agent, label, carbohydrate, carrier,immunoglobulin or immunoglobulin fragment, or an immunomodulatory agent.In another embodiment, the carbohydrate may be mannose, fucose, glucose,GlcNAs, or maltose. In another embodiment, the carrier may be KeyholeLimpit Hemocyannin (KLH), diphtheria toxoid, cholera toxoid, ovalbumin,bovine serum albumin (BSA), Pseudomonas exoprotein A, or microbial outermembrane proteins (OMPS). In a further embodiment, the conjugate maycomprise a polypeptide comprising the amino acid sequence ofFPEDYFRYTNQK (SEQ ID NO: 4) conjugated to KLH. In a further embodiment,the conjugate may comprise a polypeptide comprising the amino acidsequence of SLPDDWFRYINY (SEQ ID NO: 5) conjugated to KLH. In anotherembodiment, the label may be a chemiluminescent label, paramagneticlabel, MRI contrast agent, fluorescent label, bioluminescent label, orradioactive label. In another embodiment, the paramagnetic label may bealuminum, manganese, platinum, oxygen, lanthanum, lutetium, scandium,yttrium, or gallium. In another embodiment, the cytotoxic agent may be amoiety that inhibits DNA, RNA, or protein synthesis, a radionuclide, orribosomal inhibiting protein. In another embodiment, the cytotoxic agentmay be ²¹²Bi, ¹³¹I, ¹⁸⁸Re, ⁹⁰Y, vindesine, methotrexate, adriamycin,cisplatin, pokeweed antiviral protein, Pseudomonas exotoxin A, ricin,diphtheria toxin, ricin A chain, or cytotoxic phospholipase enzyme.

In one embodiment, the invention provides a composition comprising thepolypeptide of any one of the amino acid sequences of SEQ ID NO: 1-24,or combinations thereof. In another embodiment, the composition furthercomprises a pharmaceutically acceptable carrier, diagnosticallyacceptable carrier, adjuvant, or excipient. In a further embodiment, thecomposition may comprise the polypeptide comprising the amino acidsequence of SEQ ID NO: 4. In a further embodiment, the composition maycomprise the polypeptide comprising the amino acid sequence of SEQ IDNO: 5. In one embodiment, the composition may be a pharmaceuticalcomposition, an antigenic composition, or an immunogenic composition. Inanother embodiment, the invention provides a diagnostic kit comprisingthe polypeptide of any one of the amino acid sequences of SEQ ID NO:1-24, or combinations thereof. In another embodiment, the polypeptide ofthe kit is directly or indirectly attached to a solid phase support orcell membrane. In another embodiment, the solid phase support may be abead, plate, matrix, polymer, test tube, sheet, culture dish, or teststrip. In another embodiment, the solid phase support may be an array.In another embodiment, the polypeptide may be at least about 80% or 90%identical to any one of the amino acid sequences of SEQ ID NO: 1-24, orcombinations thereof.

In one embodiment, the invention provides a composition comprising afusion protein comprising the polypeptide of any one of the amino acidsequences of SEQ ID NO: 1-24, or combinations thereof. In anotherembodiment, the composition further comprises a pharmaceuticallyacceptable carrier, diagnostically acceptable carrier, adjuvant, orexcipient. In one embodiment, the composition may be a pharmaceuticalcomposition, an antigenic composition, or an immunogenic composition. Inanother embodiment, the invention provides a diagnostic kit comprising afusion protein comprising the polypeptide of any one of the amino acidsequences of SEQ ID NO: 1-24, or combinations thereof. In anotherembodiment, the polypeptide of the kit is directly or indirectlyattached to a solid phase support or cell membrane. In anotherembodiment, the solid phase support may be a bead, plate, matrix,polymer, test tube, sheet, culture dish, or test strip. In anotherembodiment, the solid phase support may be an array. In anotherembodiment, the polypeptide may be at least about 80% or 90% Identicalto any one of the amino acid sequences of SEQ ID NO: 1-24, orcombinations thereof.

In one embodiment, the invention provides a composition comprising aconjugate comprising the polypeptide of any one of the amino acidsequences of SEQ ID NO: 1-24, or combinations thereof. In anotherembodiment, the composition further comprises a pharmaceuticallyacceptable carrier, diagnostically acceptable carrier, adjuvant, orexcipient. In one embodiment, the composition may be a pharmaceuticalcomposition, an antigenic composition, or an immunogenic composition. Inanother embodiment, the invention provides a diagnostic kit comprising aconjugate comprising the polypeptide of any one of the amino acidsequences of SEQ ID NO: 1-24, or combinations thereof. In anotherembodiment, the polypeptide of the kit is directly or indirectlyattached to a solid phase support or cell membrane. In anotherembodiment, the solid phase support may be a bead, plate, matrix,polymer, test tube, sheet, culture dish, or test strip. In anotherembodiment, the solid phase support may be an array. In anotherembodiment, the polypeptide may be at least about 80% or 90% identicalto any one of the amino acid sequences of SEQ ID NO: 1-24, orcombinations thereof.

In one embodiment, the invention provides an isolated polynucleotidethat encodes the polypeptide of any one of the amino acid sequences ofSEQ ID NO: 1-24. In another embodiment, the invention provides anisolated expression vector comprising an isolated polynucleotide thatencodes the polypeptide of any one of the amino acid sequences of SEQ IDNO: 1-24. In another embodiment, an isolated host cell comprises anisolated expression vector comprising an isolated polynucleotide thatencodes the polypeptide of any one of the amino acid sequences of SEQ IDNO: 1-24. In a further embodiment, a non-human transgenic animal maycomprise a host cell comprises an expression vector comprising anisolated polynucleotide that encodes the polypeptide of any one of theamino acid sequences of SEQ ID NO: 1-24. The invention also provides fora composition that may comprise an isolated polynucleotide that encodesthe polypeptide of any one of the amino acid sequences of SEQ ID NO:1-24. In another embodiment, the composition may further comprise apharmaceutically acceptable carrier, adjuvant, or excipient. In anotherembodiment, the polypeptide may be at least about 80% or 90% identicalto any one of the amino acid sequences of SEQ ID NO: 1-24, orcombinations thereof.

In one embodiment, the invention provides an isolated polynucleotidethat encodes a fusion protein comprising a polypeptide comprising anyone of the amino acid sequences of SEQ ID NO: 1-24. In anotherembodiment, the invention provides an isolated expression vectorcomprising an isolated polynucleotide that encodes a fusion proteincomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In another embodiment, an isolated host cellcomprises an isolated expression vector comprising an isolatedpolynucleotide that encodes a fusion protein comprising a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24. In afurther embodiment, a non-human transgenic animal may comprise a hostcell comprises an expression vector comprising an isolatedpolynucleotide that encodes a fusion protein comprising a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24. Theinvention also provides for a composition that may comprise an isolatedpolynucleotide that encodes a fusion protein comprising a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24. Inanother embodiment, the composition may further comprise apharmaceutically acceptable carrier, adjuvant, or excipient. In anotherembodiment, the polypeptide may be at least about 80% or 90% identicalto any one of the amino acid sequences of SEQ ID NO: 1-24, orcombinations thereof.

The invention also provides for an isolated antibody or anantigen-binding fragment thereof that selectively binds a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24. Theinvention also provides for an isolated antibody or an antigen-bindingfragment thereof that selectively binds a fusion protein comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24. The invention also provides for an isolated antibody or anantigen-binding fragment thereof that selectively binds a conjugatecomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In another embodiment, the antibody orantigen-binding fragment thereof may be produced using a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24, afusion protein comprising a polypeptide comprising any one of the aminoacid sequences of SEQ ID NO: 1-24, or a conjugate comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24 as the immunogen. In another embodiment, the polypeptide may be atleast about 80% or 90% identical to any one of the amino acid sequencesof SEQ ID NO: 1-24, or combinations thereof.

In another embodiment, the antibody or antigen-binding fragment thereofmay be recombinant. In another embodiment, the antibody orantigen-binding fragment thereof may have anti-tumor activity. In oneembodiment, the fragment may be a Fab, Fab′, F(ab′)2, Fv, CDR, paratope,or portion of an antibody that is capable of binding the antigen. Inanother embodiment, the antibody or antigen-binding fragment thereof maybe chimeric, humanized, anti-idiotypic, single-chain, bifunctional, orco-specific. In one embodiment, the antibody or antigen-binding fragmentthereof may be directly or indirectly conjugated to a label, cytotoxicagent, therapeutic agent, or an immunosuppressive agent. In oneembodiment, the label may be a chemiluminescent label, paramagneticlabel, MRI contrast agent, fluorescent label, bioluminescent label, orradioactive label. In one embodiment, the paramagnetic label may bealuminum, manganese, platinum, oxygen, lanthanum, lutetium, scandium,yttrium, or gallium. In one embodiment, the cytotoxic agent may be amoiety that inhibits DNA, RNA, or protein synthesis, a radionuclide, orribosomal inhibiting protein, ²¹²Bi, ¹³¹I, ¹⁸⁸Re, ⁹⁰Y, vindesine,methotrexate, adriamycin, cisplatin, pokeweed antiviral protein,Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, orcytotoxic phospholipase enzyme. In one embodiment, the therapeutic agentmay be a lymphokine or growth factor, growth factor receptor, TollReceptor or an agonist or antagonist of any of the foregoing. In oneembodiment, the immunmodulatory agent may be an immunosuppressive agentselected from a cyclosporine, leflunomide, methotrexate, azothiprine,mercaptopurine, dactinomycin, tacrolimus, or sirolimus.

The invention also provides a composition comprising an antibody orantibody fragment which selectively binds a polypeptide comprising anyone of the amino acid sequences of SEQ ID NO: 1-24, a fusion proteincomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24, or a conjugate comprising a polypeptide comprisingany one of the amino acid sequences of SEQ ID NO: 1-24. In anotherembodiment, the composition may further comprise a pharmaceuticallyacceptable carrier, adjuvant, or excipient. In another embodiment, thepolypeptide may be at least about 80% or 90% identical to any one of theamino acid sequences of SEQ ID NO: 1-24, or combinations thereof. Adiagnostic kit comprising an antibody or antibody fragment whichselectively binds a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24, a fusion protein comprising a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24, or aconjugate comprising a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24. In another embodiment, the polypeptide ofthe kit is directly or indirectly attached to a solid phase support orcell membrane. In another embodiment, the solid phase support may be abead, plate, matrix, polymer, test tube, sheet, culture dish, or teststrip. In another embodiment, the solid phase support may be an array.

In another embodiment, the invention provides a composition for treatingcancer comprising an effective amount of the polypeptide a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24. In afurther embodiment, the composition for treating cancer may comprise aneffective amount of a fusion protein comprising a polypeptide comprisingany one of the amino acid sequences of SEQ ID NO: 1-24. In a furtherembodiment, the composition for treating cancer may comprise aneffective amount of a conjugate comprising a polypeptide comprising anyone of the amino acid sequences of SEQ ID NO: 1-24. In a furtherembodiment, the composition for treating cancer may comprise aneffective amount of an antibody or antibody fragment which selectivelybinds a polypeptide comprising any one of the amino acid sequences ofSEQ ID NO: 1-24, a fusion protein comprising a polypeptide comprisingany one of the amino acid sequences of SEQ ID NO: 1-24, or a conjugatecomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In another embodiment, the cancer may be selectedfrom the group consisting of lung, breast, ovarian, stomach, pancreas,uterine, esophageal, colorectal, and liver cancer. In anotherembodiment, the cancer is pancreas or colorectal cancer.

The invention also provides for the use of a polypeptide comprising anyone of the amino acid sequences of SEQ ID NO: 1-24 in the preparation ofa medicament for treating cancer. In another embodiment, the use of afusion protein comprising a polypeptide comprising any one of the aminoacid sequences of SEQ ID NO: 1-24 in the preparation of a medicament fortreating cancer. In a further embodiment, use of a conjugate comprisinga polypeptide comprising any one of the amino acid sequences of SEQ IDNO: 1-24 in the preparation of a medicament for treating cancer. In afurther embodiment, use of an antibody or antibody fragment whichselectively binds a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24, a fusion protein comprising a polypeptidecomprising any one of the amino acid sequences of SEQ ID NO: 1-24, or aconjugate comprising a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24 in the preparation of a medicament fortreating cancer. In another embodiment, the cancer may be selected fromthe group consisting of lung, breast, ovarian, stomach, pancreas,uterine, esophageal, colorectal, and liver cancer. In anotherembodiment, the cancer is pancreas or colorectal cancer.

The invention also provides a method for treating cancer may compriseadministering an effective amount of a polypeptide comprising any one ofthe amino acid sequences of SEQ ID NO: 1-24. In another embodiment, amethod for slowing the growth of a tumor may comprise administering aneffective amount of a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24. In one embodiment, a method for promotingtumor regression in a subject may comprise administering an effectiveamount of a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In one embodiment, a method for activating dendriticcells may comprise removing dendritic cells from a patient, contactingcells ex vivo with an effective amount of a polypeptide comprising anyone of the amino acid sequences of SEQ ID NO: 1-24, and reintroducingactivated the dendritic cells into said patient. In one embodiment, amethod for activating antigen-specific immunity may compriseadministering an effective amount of a polypeptide comprising any one ofthe amino acid sequences of SEQ ID NO: 1-24.

The invention also provides a method for treating cancer may compriseadministering an effective amount of a fusion protein comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24. In one embodiment, a method for slowing the growth of a tumor maycomprise administering an effective amount of a fusion proteincomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In one embodiment, a method for promoting tumorregression in a subject may comprise administering an effective amountof a fusion protein comprising a polypeptide comprising any one of theamino acid sequences of SEQ ID NO: 1-24. In one embodiment, a method foractivating dendritic cells may comprise removing dendritic cells from apatient, contacting cells ex vivo with an effective amount of a fusionprotein comprising a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24, and reintroducing activated the dendriticcells into said patient. In one embodiment, a method for activatingantigen-specific immunity may comprise administering an effective amountof a fusion protein comprising a polypeptide comprising any one of theamino acid sequences of SEQ ID NO: 1-24.

The invention also provides a method for treating cancer may compriseadministering an effective amount of a conjugate comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24. In one embodiment, a method for slowing the growth of a tumor maycomprise administering an effective amount of a conjugate comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24. In one embodiment, a method for promoting tumor regression in asubject may comprise administering an effective amount of a conjugatecomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In one embodiment, a method for activating dendriticcells may comprise removing dendritic cells from a patient, contactingcells ex vivo with an effective amount of a conjugate comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24, and reintroducing activated the dendritic cells into said patient.In one embodiment, a method for activating antigen-specific immunity maycomprise administering an effective amount of a fusion protein orconjugate comprising a polypeptide comprising any one of the amino acidsequences of SEQ ID NO: 1-24.

The invention further provides a method for treating cancer comprisingadministering an effective amount of an antibody or antibody fragmentwhich selectively binds a polypeptide comprising any one of the aminoacid sequences of SEQ ID NO: 1-24, a fusion protein comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24, or a conjugate comprising a polypeptide comprising any one of theamino acid sequences of SEQ ID NO: 1-24. In one embodiment, a method forslowing the growth of a tumor and/or inhibiting metastasis may compriseadministering an effective amount of an antibody or antibody fragmentwhich selectively binds a polypeptide comprising any one of the aminoacid sequences of SEQ ID NO: 1-24, a fusion protein comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24, or a conjugate comprising a polypeptide comprising any one of theamino acid sequences of SEQ ID NO: 1-24. In one embodiment, a method forpromoting tumor regression in a subject may comprise administering aneffective amount of an antibody or antibody fragment which selectivelybinds a polypeptide comprising any one of the amino acid sequences ofSEQ ID NO: 1-24, a fusion protein comprising a polypeptide comprisingany one of the amino acid sequences of SEQ ID NO: 1-24, or a conjugatecomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24. In one embodiment, a method for activating dendriticcells may comprise removing dendritic cells from a patient, contactingcells ex vivo with an effective amount of an antibody or antibodyfragment which selectively binds a polypeptide comprising any one of theamino acid sequences of SEQ ID NO: 1-24, a fusion protein comprising apolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-24, or a conjugate comprising a polypeptide comprising any one of theamino acid sequences of SEQ ID NO: 1-24, and reintroducing activated thedendritic cells into said patient. In one embodiment, a method foractivating antigen-specific immunity may comprise administering aneffective amount of an antibody or antibody fragment which selectivelybinds a polypeptide comprising any one of the amino acid sequences ofSEQ ID NO: 1-24, a fusion protein comprising a polypeptide comprisingany one of the amino acid sequences of SEQ ID NO: 1-24, or a conjugatecomprising a polypeptide comprising any one of the amino acid sequencesof SEQ ID NO: 1-24.

In one embodiment, the invention provides a method for detecting a NPC-1epitope comprising: contacting a test sample with an antibody, orfragment thereof, that binds a polypeptide of any one amino acidsequence of SEQ ID NO: 1-26, and assaying for antibody-epitopecomplexes, wherein the presence of said epitope is indicative of acarcinoma. In another embodiment, the invention provides a method fordetecting the presence of a NPC-1 epitope in a patient comprisingadministering to said patient a labeled monoclonal antibody, orantigen-binding fragment thereof, that binds a polypeptide of any one ofany one amino acid sequence of SEQ ID NO: 1-26, and detecting thepresence of a NPC-1 epitope, wherein the presence of said epitope isindicative of a carcinoma.

In another embodiment, the method may comprise imaging the NPC-1epitope. In a further embodiment, the imaging may be selected from thegroup consisting of positron emission tomography (PET), CCD low-lightmonitoring system, x-ray, CT scanning, scintigraphy, photo acousticimaging, single photon emission computed tomography (SPECT), magneticresonance imaging (MRI), ultrasound, paramagnetic imaging, andendoscopic optical coherence tomography.

In another embodiment, the polypeptide, fusion protein, conjugate, orantibody may be administered in combination with another antibody, alymphokine, or a hematopoietic growth factor. In another embodiment, theagent may be administered simultaneously or sequentially with theantibody. In another embodiment, the cancer may be lung, breast,ovarian, stomach, pancreas, uterine, esophageal, colorectal, or livercancer. In a further embodiment, the cancer is pancreas or colorectalcancer. In a still further embodiment, the cancer may be pancreascancer. In another embodiment, the cancer is colorectal cancer.

In another embodiment, the cancer may be a stage 1, 2, 3 or 4 cancer. Inanother embodiment, the cancer may have metastasized. In anotherembodiment, the patient may express detectable levels of a NPC-1epitope. In another embodiment, the antigen may be detected in a tumorbiopsy sample or in the blood, stool, urine, or lymph fluid. In anotherembodiment, the patient may be at risk of cancer. In another embodiment,the patient may be a patient without symptoms.

In one embodiment, the antibody or antigen-binding fragment thereof maybe recombinant. In another embodiment, the antibody or antigen-bindingfragment thereof may have anti-tumor activity. In a further embodiment,the antigen-binding fragment thereof may be a Fab, Fab′, F(ab′)2, Fv,CDR, paratope, or portion of an antibody that is capable of binding theantigen. In another embodiment, the antibody may be chimeric, humanized,anti-idiotypic, single-chain, bifunctional, or co-specific.

In one embodiment, the antibody or antigen-binding fragment may beconjugated to a label. In one embodiment, the label may be achemiluminescent label, paramagnetic label, an MRI contrast agent,fluorescent label, bioluminescent label, or radioactive label. In oneembodiment, the paramagnetic label may be aluminum, manganese, platinum,oxygen, lanthanum, lutetium, scandium, yttrium, or gallium.

In a further embodiment, the antibody may be attached to a solidsupport. In a further embodiment, the solid support may be a bead, testtube, sheet, culture dish, or test strip. In a further embodiment, thesolid support may be an army. In a further embodiment, the sample may bea tissue biopsy, lymph, urine, cerebrospinal fluid, amniotic fluid,inflammatory exudate, blood, serum, stool, or liquid collected from thecolorectal tract.

In a further embodiment, the antibody-epitope complex may be detected byan assay selected from the group consisting of Western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitation reactions, geldiffusion precipitation reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunohistochemical assays,fluorescent immunoassays, and protein A immunoassays.

In a further embodiment, the method may detect colorectal polyps. In oneembodiment, the method may further comprise additional testing for thepresence of tumors. In a further embodiment, the method may detectbenign tumors. In a further embodiment, the method may malignant tumors.

In a further embodiment, the method may metastatic tumors. In a furtherembodiment, the method may non-metastatic tumors. In another embodiment,the method may detect pre-cancerous cells that express a cell markercomprising a NPC-1 epitope. In another embodiment, the test sample maybe obtained from a patient at risk of cancer. In another embodiment, thetest sample may be obtained from a patient without symptoms.

In one embodiment, the invention provides a method of making antibodiescomprising: immunizing an animal with a polypeptide of any one of theamino acid sequences of SEQ ID NOs: 1-24, removing said animal's spleenand prepare a single cell suspension, fusing a spleen cell with amyeloma cell, culturing post-fusion cells in hybridoma selection medium,culturing the resultant hybridomas, screening for specific antibodyproduction, and selecting hybridomas which produce the desired antibody.In another embodiment, the invention provides a method of makingantibodies comprising: immunizing an animal with a polypeptide ofFPEDYFRYTNQK (SEQ ID NO: 4) or SLPDDWFRYINY (SEQ ID NO: 5), removingsaid animal's spleen and prepare a single cell suspension, fusing aspleen cell with a myeloma cell, culturing post-fusion cells inhybridoma selection medium, culturing the resultant hybridomas,screening for specific antibody production, and selecting hybridomaswhich produce the desired antibody.

In one embodiment, the invention provides a method of making antibodiescomprising: immunizing an animal with a fusion protein comprising apolypeptide of any one of the amino acid sequences of SEQ ID NOs: 1-24,removing said animal's spleen and prepare a single cell suspension,fusing a spleen cell with a myeloma cell, culturing post-fusion cells inhybridoma selection medium, culturing the resultant hybridomas,screening for specific antibody production, and selecting hybridomaswhich produce the desired antibody. In another embodiment, the inventionprovides a method of making antibodies comprising: immunizing an animalwith a fusion protein comprising a polypeptide of FPEDYFRYTNQK (SEQ IDNO: 4) or SLPDDWFRYINY (SEQ ID NO: 5), removing said animal's spleen andprepare a single cell suspension, fusing a spleen cell with a myelomacell, culturing post-fusion cells in hybridoma selection medium,culturing the resultant hybridomas, screening for specific antibodyproduction, and selecting hybridomas which produce the desired antibody.

In one embodiment, the invention provides a method of making antibodiescomprising: immunizing an animal with a conjugate comprising apolypeptide of any one of the amino acid sequences of SEQ ID NOs: 1-24,removing said animal's spleen and prepare a single cell suspension,fusing a spleen cell with a myeloma cell, culturing post-fusion cells inhybridoma selection medium, culturing the resultant hybridomas,screening for specific antibody production, and selecting hybridomaswhich produce the desired antibody. In another embodiment, the inventionprovides a method of making antibodies comprising: immunizing an animalwith a conjugate comprising a polypeptide of FPEDYFRYTNQK (SEQ ID NO: 4)or SLPDDWFRYINY (SEQ ID NO: 5), removing said animal's spleen andprepare a single cell suspension, fusing a spleen cell with a myelomacell, culturing post-fusion cells in hybridoma selection medium,culturing the resultant hybridomas, screening for specific antibodyproduction, and selecting hybridomas which produce the desired antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of detecting NPC-1 antigen in stool frompatients with a normal colonoscopy result, small polyps (SP), multiplepolyps (MP), large polyps (LP), and colon cancer (CC). The results shownin FIG. 1 suggests a correlation between the level of NPC-1 antigendetecting in a stool sample with the presence of polyps and/or coloncancer, where the higher the NPC-1 antigen amount detected is correlatedthe larger the polyps or the higher the number of polyps. Further, thedata is suggestive of the high levels of NPC-1 antigen (e.g., over20,000 units of NPC-1 antigen in a sample) as indicative of coloncancer.

FIG. 2A-B depicts the detection of NPC-1 antigen in cancer patientsusing a NEO-101 antibody. FIG. 2A depicts a scatter plot of NPC-1antigen detection in cancer patients undergoing treatment at 1 month, 2months, and 3 months compared to normal controls. Serial blood draws ofcancer patients over an approximate 3 month period were tested. TheNEO-101 sandwich ELISA was performed at a 1:24 serum dilution. Resultsare presented as a scatter plot of each experimental group, with themean and standard error of the mean. There were 28 normal sera, 41colon/pancreas cancer sera at 1-month, 33 colon/pancreas cancer sera at2-month, and 25 colon/pancreas cancer sera at 3-month. FIG. 2B depicts ascatter plot showing that colorectal and pancreas cancer sera aredetected similarly by NEO-101. Serum specimens were sorted according topatients diagnosed with either colorectal (n=36) or pancreas cancer(n=5). These were compared to the average of all cancer specimens andthe normal serum specimens

FIG. 3 depicts anti-tumor activity in human AsPC-1 pancreas tumorxenograft model in nude mice comparing administration of saline, humanIgG (200 μg), and NEO-101 (200 μg) comprising two cycles of treatment.The heavy arrows indicate days of NEO-101 injection (ip), light arrowsindicate days of PBMC injection (ip), the asterisk (*) indicatesstatistically significant differences between NEO-101 treated mice withhuman IgG treated mice.

FIG. 4 depicts anti-tumor activity in human AsPC-1 pancreas tumorxenograft model in nude mice comparing administration of saline, humanIgG (200 μg), and NEO-101 (200 μg) where four cycles of treatment wereadministered instead of two cycles. The heavy arrows indicate days ofNEO-101 injection (ip), light arrows indicate days of PBMC injection(ip), the asterisk (*) indicates statistically significant differencesbetween NEO-101 treated mice with human IgG treated mice.

FIG. 5 depicts anti-tumor activity in human LS174T colorectal tumorxenograft model in nude mice comparing administration of saline, humanIgG (200 μg), and NEO-101 (200 μg). The heavy arrows indicate days ofNEO-01 injection (ip), light arrows indicate days of PBMC injection(ip), the asterisk (*) indicates statistically significant differencesbetween NEO-101 treated mice with human IgG treated mice.

FIG. 6 depicts the results of peptide sequencing following severalrounds of phage library biopanning identified using NEO-101 antibody and4B6 anti-idiotypic antibody.

FIG. 7 is a bar graph depicting results of phage clones in NEO-101ELISA.

FIG. 8A-B depicts NEO-10 μl binding inhibition by phage M13 clones. FIG.8A depicts NEO-101 the percent binding inhibition by phage M13 clones.M13 clones were diluted 1:30 and competed with NEO-101-biotin (250ng/ml) on colon cancer antigen (3 μg/ml) coated plates. In FIG. 8B,Inhibition %=[OD of NEO-101-biotin (250 ng/ml)−OD of NEO-101-biotin (250ng/ml)+1:30 diluted M13]+OD of NEO-101-biotin (250 ng/ml). FIG. 9B showsNEO-101 binding inhibition by M13 clones on colon antigen (3μg/ml)-coated plates.

FIG. 9A depicts the percent NPC-1C binding inhibition by M13 clones inELISA. The M13 clones were derived from the biopanning that yielded 10¹¹pfu/10 μl. FIG. 9B is a bar graph depicting NPC-1C beads bindinginhibition (%) by cloned phage in beads assay.

FIG. 10 depicts peptide binding of NEO-101 in an ELISA assay.

FIG. 11 depicts peptide-biotin binding to NEO-101.

FIG. 12 is a bar graph depicting the percent inhibition of peptides toNEO-101 binding.

FIG. 13 depicts a comparison of the amino acid sequences of the NPC-1short antigen with the 4-1-3-C9 and 4-1-4-C12 peptides.

FIG. 14A depicts the binding of CFPAC1 cells by antibody coupled beads,as a percent inhibition of NEO-101 beads binding to CFPAC1 cells(rosetted cells) inhibited by the peptides 4-1-4-C12 and 4-1-4C12-R2.FIG. 14B is a bar graph showing the inhibition by 4B6 of NEO-101 beadsbinding to CFPAC1 cells (rosetted cells).

FIG. 15A is a bar graph showing the percent blocking of NEO-101 bypeptides (identified by biopanning an M13 phage library) on CFPAC1culture supernatant-coated plates. FIG. 15B is a bar graph of OD valuesshowing blocking of NEO-101 by peptides (identified by biopanning an M13phage library) on CFPAC1 culture supernatant-coated plates.

FIG. 16A-B depicts 4-1-4C12-KLH immunized serum binding to 4-1-4C12 (SEQID NO: 5) (A) and CFPAC1 human pancreatic cell line supernatant (B) in adose dependent manner.

FIG. 17 depicts that anti 4-1-4C12 pAb binds to CFPAC1 human pancreaticcell line supernatant, BSM and 4-1-4C12 peptide in a dose dependentmanner.

FIG. 18 depicts that anti 4-1-4C12 pAb has lower affinity to BSM whencompared with NPC-1C antibody in binding ELISA.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be fully understood,the following detailed description is set forth. Various embodiments ofthe invention are described in detail and may be further illustrated bythe provided examples.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein may be used inthe invention or testing of the present invention, suitable methods andmaterials are described herein. The materials, methods and examples areillustrative only, and are not intended to be limiting.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

“Adjuvant,” as used herein, refers broadly to any substance which isincorporated into or administered simultaneously with NPC-1 epitopepeptidomimetic of the invention which potentiates the immune response inthe subject. Adjuvants include but are not limited to aluminumcompounds, e.g., gels, aluminum hydroxide and aluminum phosphate, andFreund's complete or incomplete adjuvant (e.g., in which the PS/Aantigen is incorporated in the aqueous phase of a stabilized water inparaffin oil emulsion). The paraffin oil may be replaced with differenttypes of oils, e.g., squalene or peanut oil. Other materials withadjuvant properties, include BCG (attenuated Mycobacteriumtuberculosis), calcium phosphate, levamisole, isoprinosine, polyanions(e.g., poly A:U), lentinan, pertussis toxin, lipid A, saponins, QS-21and peptides, e.g. muramyl dipeptide. Rare earth salts, e.g., lanthanumand cerium, may also be used as adjuvants. The amount of adjuvantsdepends on the subject and the particular antigen used and can bereadily determined by one skilled in the art without undueexperimentation.

“Amino acid,” as used herein, refers broadly to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

“Antibody,” as used herein, refers broadly to any polypeptidechain-containing molecular structure with a specific shape that fits toand recognizes an epitope, where one or more non-covalent bindinginteractions stabilize the complex between the molecular structure andthe epitope. The archetypal antibody molecule is the immunoglobulin, andall types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, from all sources,e.g., human, rodent, rabbit, cow, sheep, pig, dog, chicken, areconsidered to be “antibodies.” Antibodies include but are not limited tochimeric antibodies, human antibodies and other non-human mammalianantibodies, humanized antibodies, single chain antibodies (scFvs),camelbodies, nanobodies, IgNAR (single-chain antibodies derived fromsharks), small-modular immunopharmaceuticals (SMIPs), and antibodyfragments (e.g., Fabs, Fab′, F(ab′)₂.) Numerous antibody codingsequences have been described; and others may be raised by methodswell-known in the art. See Streltsov, et al. (2005) Protein Sci. 14(11):2901-9; Greenberg, et al. (1995) Nature 374(6518): 168-173; Nuttall, etaL (2001) Mol Immunol. 38(4): 313-26; Hamers-Casterman, et al. (1993)Nature 363(6428): 446-8; Gill, et al. (2006) Curr Opin Biotechnol.17(6): 653-8.

“Antigen,” as used herein, refers broadly to a molecule or a portion ofa molecule capable of being bound by an antibody which is additionallycapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen may have one epitope, or havemore than one epitope. The specific reaction referred to hereinindicates that the antigen will react, in a highly selective manner,with its corresponding antibody and not with the multitude of otherantibodies which may be evoked by other antigens. Antigens may be tumorspecific (e.g., expressed by neoplastic cells of pancreatic and coloncarcinoma.)

“Antigenic composition,” as used herein, refers broadly to a compositionthat elicits an immune response.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor.

“Chimeric antibody,” as used herein, refers broadly to an antibodymolecule in which the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, or the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

“Conservatively modified variants,” as used herein, applies to bothamino acid and nucleic acid sequences, and with respect to particularnucleic acid sequences, refers broadly to conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Suchnucleic acid variations are “silent variations,” which are one speciesof conservatively modified variations. Every nucleic acid sequenceherein which encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) may be modified to yield a functionally identical molecule.

“Complementarity determining region,” “hypervariable region,” or “CDR,”as used herein, refers broadly to one or more of the hyper-variable orcomplementarily determining regions (CDRs) found in the variable regionsof light or heavy chains of an antibody. See Kabat, et al. (1987)“Sequences of Proteins of Immunological Interest” National Institutes ofHealth, Bethesda, Md. These expressions include the hypervariableregions as defined by Kabat, et al. (1983) “Sequences of Proteins ofImmunological Interest” U.S. Dept. of Health and Human Services or thehypervariable loops in 3-dimensional structures of antibodies. Chothiaand Lesk (1987) J Mol. Biol. 196: 901-917. The CDRs in each chain areheld in close proximity by framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen binding site.Within the CDRs there are select amino acids that have been described asthe selectivity determining regions (SDRs) which represent the criticalcontact residues used by the CDR in the antibody-antigen interaction.Kashmiri (2005) Methods 36: 25-34.

“Control amount,” as used herein, refers broadly to a marker can be anyamount or a range of amounts to be compared against a test amount of amarker. For example, a control amount of a marker may be the amount of amarker in a patient with a particular disease or condition or a personwithout such a disease or condition. A control amount can be either inabsolute amount (e.g., microgram/ml) or a relative amount (e.g.,relative intensity of signals).

“Differentially present,” as used herein, refers broadly to differencesin the quantity or quality of a marker present in a sample taken frompatients having a disease or condition as compared to a comparablesample taken from patients who do not have one of the diseases orconditions. For example, a nucleic acid fragment may optionally bedifferentially present between the two samples if the amount of thenucleic acid fragment in one sample is significantly different from theamount of the nucleic acid fragment in the other sample, for example asmeasured by hybridization and/or NAT-based assays. A polypeptide isdifferentially present between the two samples if the amount of thepolypeptide in one sample is significantly different from the amount ofthe polypeptide in the other sample. It should be noted that if themarker is detectable in one sample and not detectable in the other, thensuch a marker may be considered to be differentially present.Optionally, a relatively low amount of up-regulation may serve as themarker.

“Diagnostic,” as used herein, refers broadly to identifying the presenceor nature of a pathologic condition. Diagnostic methods differ in theirsensitivity and specificity. The “sensitivity” of a diagnostic assay isthe percentage of diseased individuals who test positive (percent of“true positives”). Diseased individuals not detected by the assay are“false negatives.” Subjects who are not diseased and who test negativein the assay are termed “true negatives.” The “specificity” of adiagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

“Diagnosing,” as used herein, refers broadly to classifying a disease ora symptom, determining a severity of the disease, monitoring diseaseprogression, forecasting an outcome of a disease and/or prospects ofrecovery. The term “detecting” may also optionally encompass any of theforegoing. Diagnosis of a disease according to the present inventionmay, in some embodiments, be affected by determining a level of apolynucleotide or a polypeptide of the present invention in a biologicalsample obtained from the subject, wherein the level determined can becorrelated with predisposition to, or presence or absence of thedisease. It should be noted that a “biological sample obtained from thesubject” may also optionally comprise a sample that has not beenphysically removed from the subject.

“Effective amount,” as used herein, refers broadly to the amount of acompound, antibody, antigen, or cells that, when administered to apatient for treating a disease, is sufficient to effect such treatmentfor the disease. The effective amount may be an amount effective forprophylaxis, and/or an amount effective for prevention. The effectiveamount may be an amount effective to reduce, an amount effective toprevent the incidence of signs/symptoms, to reduce the severity of theincidence of signs/symptoms, to eliminate the incidence ofsigns/symptoms, to slow the development of the incidence ofsigns/symptoms, to prevent the development of the incidence ofsigns/symptoms, and/or effect prophylaxis of the incidence ofsigns/symptoms. The “effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, susceptibility,and pre-existing conditions, of the patient to be treated. The term“effective amount” is synonymous with “therapeutically effective amount”for purposes of this invention.

“Expression vector,” as used herein, refers broadly to any recombinantexpression system for the purpose of expressing a nucleic acid sequenceof the invention in vitro or in vivo, constitutively or inducibly, inany cell, including prokaryotic, yeast, fungal, plant, insect ormammalian cell. The term includes linear or circular expression systems.The term includes expression systems that remain episomal or integrateinto the host cell genome. The expression systems can have the abilityto self-replicate or not, i.e., drive only transient expression in acell. The term includes recombinant expression cassettes which containonly the minimum elements needed for transcription of the recombinantnucleic acid.

“Framework region” or “FR,” as used herein, refers broadly to one ormore of the framework regions within the variable regions of the lightand heavy chains of an antibody. See Kabat, et al. (1987) “Sequences ofProteins of Immunological Interest.” National Institutes of Health,Bethesda, Md. These expressions include those amino acid sequenceregions interposed between the CDRs within the variable regions of thelight and heavy chains of an antibody.

“Heterologous,” as used herein, refers broadly to portions of a nucleicacid indicates that the nucleic acid comprises two or more subsequencesthat are not found in the same relationship to each other in nature. Forinstance, the nucleic acid is typically recombinantly produced, havingtwo or more sequences from unrelated genes arranged to make a newfunctional nucleic acid, e.g., a promoter from one source and a codingregion from another source. Similarly, a heterologous protein indicatesthat the protein comprises two or more subsequences that are not foundin the same relationship to each other in nature (e.g., a fusionprotein).

“High affinity,” as used herein, refers broadly to an antibody having aKD of at least 10⁻⁸ M, more preferably at least 10⁻⁹ M and even morepreferably at least 10⁻¹⁰ M for a target antigen. However, “highaffinity” binding can vary for other antibody isotypes. For example,“high affinity” binding for an IgM isotype refers to an antibody havinga KD of at least 10⁻⁷ M, more preferably at least 10⁻⁸ M.

“Homology,” as used herein, refers broadly to a degree of similaritybetween a nucleic acid sequence and a reference nucleic acid sequence orbetween a polypeptide sequence and a reference polypeptide sequence.Homology may be partial or complete. Complete homology indicates thatthe nucleic acid or amino acid sequences are identical. A partiallyhomologous nucleic acid or amino acid sequence is one that is notidentical to the reference nucleic acid or amino acid sequence. Thedegree of homology can be determined by sequence comparison. The term“sequence identity” may be used interchangeably with “homology.”

“Host cell,” as used herein, refers broadly to a cell that contains anexpression vector and supports the replication or expression of theexpression vector. Host cells may be prokaryotic cells such as E. coli,or eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, ormammalian cells such as CHO, HeLa, HEK-293, e.g., cultured cells,explants, and cells in vivo.

“Hybridization,” as used herein, refers broadly to the physicalinteraction of complementary (including partially complementary)polynucleotide strands by the formation of hydrogen bonds betweencomplementary nucleotides when the strands are arranged antiparallel toeach other.

“K-assoc” or “Ka”, as used herein, refers broadly to the associationrate of a particular antibody-antigen interaction, whereas the term“Kdiss” or “Kd,” as used herein, refers to the dissociation rate of aparticular antibody-antigen interaction. The term “KD”, as used herein,is intended to refer to the dissociation constant, which is obtainedfrom the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molarconcentration (M). KD values for antibodies can be determined usingmethods well established in the art.

“Immunoassay,” as used herein, refers broadly to an assay that uses anantibody to specifically bind an antigen. The immunoassay may becharacterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

“Isolated,” as used herein, refers broadly to material removed from itsoriginal environment in which it naturally occurs, and thus is alteredby the hand of man from its natural environment. Isolated material maybe, for example, exogenous nucleic acid included in a vector system,exogenous nucleic acid contained within a host cell, or any materialwhich has been removed from its original environment and thus altered bythe hand of man (e.g., “isolated antibody or isolated peptidomimetic”).

“Label” or a “detectable moiety” as used herein, refers broadly to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means.

“Low stringency,” “medium stringency,” “high stringency,” or “very highstringency conditions,” as used herein, refers broadly to conditions fornucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel, et al. (2002) ShortProtocols in Molecular Biology (5^(th) Ed.) John Wiley & Sons, NY.Exemplary specific hybridization conditions include but are not limitedto: (1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); (2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) highstringency hybridization conditions in 6×SSC at about 450C, followed byone or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and (4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

“Mammal,” as used herein, refers broadly to any and all warm-bloodedvertebrate animals of the class Mammalia, including humans,characterized by a covering of hair on the skin and, in the female,milk-producing mammary glands for nourishing the young. Examples ofmammals include but are not limited to alpacas, armadillos, capybaras,cats, camels, chimpanzees, chinchillas, cattle, dogs, gerbils, goats,gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-humanprimates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammalsinclude but are not limited to bovine, canine, equine, feline, murine,ovine, porcine, primate, and rodent species. Mammal also includes anyand all those listed on the Mammal Species of the World maintained bythe National Museum of Natural History, Smithsonian Institution inWashington D.C.

“Nucleic acid” or “nucleic acid sequence,” as used herein, refersbroadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide ineither single- or double-stranded form. The term encompasses nucleicacids, i.e., oligonucleotides, containing known analogs of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.The term nucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

“Operatively linked”, as used herein, refers broadly to when two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

“Paratope,” as used herein, refers broadly to the part of an antibodywhich recognizes an antigen (e.g., the antigen-binding site of anantibody.) Paratopes may be a small region (e.g., 15-22 amino acids) ofthe antibody's Fv region and may contain parts of the antibody's heavyand light chains. See Goldsby, et al. Antigens (Chapter 3) Immunology(5^(th) Ed.) New York: W.H. Freeman and Company, pages 57-75.

“Patient,” as used herein, refers broadly to any animal who is in needof treatment either to alleviate a disease state or to prevent theoccurrence or reoccurrence of a disease state. Also, “Patient” as usedherein, refers broadly to any animal who has risk factors, a history ofdisease, susceptibility, symptoms, signs, was previously diagnosed, isat risk for, or is a member of a patient population for a disease. Thepatient may be a clinical patient such as a human or a veterinarypatient such as a companion, domesticated, livestock, exotic, or zooanimal. The term “subject” may be used interchangeably with the term“patient”.

“Peptidomimetic,” as used herein refers broadly to a compound that canimitate or block the biological effect of a peptide on a molecularlevel. Peptidomimetics may be polymers designed to mimic a peptide, suchas peptoids and β-peptides, or may be a peptide that mimics a differentpeptide.

“Polypeptide,” “peptide” and “protein,” are used interchangeably andrefer broadly to a polymer of amino acid residues. The terms apply toamino acid polymers in which one or more amino acid residue is an analogor mimetic of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Polypeptides can be modified, e.g., by theaddition of carbohydrate residues to form glycoproteins. The terms“polypeptide,” “peptide” and “protein” include glycoproteins, as well asnon-glycoproteins.

“Promoter,” as used herein, refers broadly to an array of nucleic acidsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation.

“Prophylactically effective amount,” as used herein, refers broadly tothe amount of a compound that, when administered to a patient forprophylaxis of a disease or prevention of the reoccurrence of a disease,is sufficient to effect such prophylaxis for the disease orreoccurrence. The prophylactically effective amount may be an amounteffective to prevent the incidence of signs and/or symptoms. The“prophylactically effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, predisposition toconditions, preexisting conditions, of the patient to be treated.

“Prophylaxis,” as used herein, refers broadly to a course of therapywhere signs and/or symptoms are not present in the patient, are inremission, or were previously present in a patient. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient. Further, prevention includes treating patients who maypotentially develop the disease, especially patients who are susceptibleto the disease (e.g., members of a patent population, those with riskfactors, or at risk for developing the disease).

“Recombinant” as used herein, refers broadly with reference to aproduct, e.g., to a cell, or nucleic acid, protein, or vector, indicatesthat the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified. Thus, for example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cellor express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

“Specifically (or selectively) binds” to an antibody or “specifically(or selectively) immunoreactive with,” or “specifically interacts orbinds,” as used herein, refers broadly to a protein or peptide (or otherepitope), refers, in some embodiments, to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. For example, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times greater than the background(non-specific signal) and do not substantially bind in a significantamount to other proteins present in the sample. Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than about 10 to 100 times background.

“Specifically hybridizable” and “complementary” as used herein, referbroadly to a nucleic acid can form hydrogen bond(s) with another nucleicacid sequence by either traditional Watson-Crick or othernon-traditional types. The binding free energy for a nucleic acidmolecule with its complementary sequence is sufficient to allow therelevant function of the nucleic acid to proceed, e.g., RNAi activity.Determination of binding free energies for nucleic acid molecules iswell known in the art. See, e.g., Turner, et al. (1987) CSH Symp. Quant.Biol. LII: 123-33; Frier, et al. (1986) PNAS 83: 9373-77; Turner, et al.(1987) J. Am. Chem. Soc. 109: 3783-85. A percent complementarityindicates the percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., about at least 5, 6, 7, 8, 9,10 out of 10 being about at least 50%, 60%, 70%, 80%, 90%, and 100%complementary, inclusive). “Perfectly complementary” or 100%complementarity refers broadly all of the contiguous residues of anucleic acid sequence hydrogen bonding with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantialcomplementarity” refers to polynucleotide strands exhibiting about atleast 90% complementarity, excluding regions of the polynucleotidestrands, such as overhangs, that are selected so as to benoncomplementary. Specific binding requires a sufficient degree ofcomplementarity to avoid non-specific binding of the oligomeric compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed. The non-targetsequences typically may differ by at least 5 nucleotides.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Solid support,” “support,” and “substrate,” as used herein, refersbroadly to any material that provides a solid or semi-solid structurewith which another material can be attached including but not limited tosmooth supports (e.g., metal, glass, plastic, silicon, and ceramicsurfaces) as well as textured and porous materials.

“Subjects” as used herein, refers broadly to anyone suitable to betreated according to the present invention include, but are not limitedto, avian and mammalian subjects, and are preferably mammalian. Mammalsof the present invention include, but are not limited to, canines,felines, bovines, caprines, equines, ovines, porcines, rodents (e.g.,rats and mice), lagomorphs, primates, humans. Any mammalian subject inneed of being treated according to the present invention is suitable.Human subjects of both genders and at any stage of development (i.e.,neonate, infant, juvenile, adolescent, adult) can be treated accordingto the present invention. The present invention may also be carried outon animal subjects, particularly mammalian subjects such as mice, rats,dogs, cats, cattle, goats, sheep, and horses for veterinary purposes,and for drug screening and drug development purposes. “Subjects” is usedinterchangeably with “patients.”

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“Therapy,” “therapeutic,” “treating,” or “treatment”, as used herein,refers broadly to treating a disease, arresting, or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, treatment, remedy, reduction,alleviation, and/or providing relief from a disease, signs, and/orsymptoms of a disease. Therapy encompasses an alleviation of signsand/or symptoms in patients with ongoing disease signs and/or symptoms(e.g., tumor growth, metastasis). Therapy also encompasses“prophylaxis”. The term “reduced”, for purpose of therapy, refersbroadly to the clinical significant reduction in signs and/or symptoms.Therapy includes treating relapses or recurrent signs and/or symptoms(e.g., tumor growth, metastasis). Therapy encompasses but is not limitedto precluding the appearance of signs and/or symptoms anytime as well asreducing existing signs and/or symptoms and eliminating existing signsand/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment Includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., tumor growth, metastasis).

“Variable region” or “VR,” as used herein, refers broadly to the domainswithin each pair of light and heavy chains in an antibody that areinvolved directly in binding the antibody to the antigen. Each heavychain has at one end a variable domain (V_(H)) followed by a number ofconstant domains. Each light chain has a variable domain (V_(L)) at oneend and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain.

“Vector,” as used herein, refers broadly to a plasmid, cosmid, phagemid,phage DNA, or other DNA molecule which is able to replicate autonomouslyin a host cell, and which is characterized by one or a small number ofrestriction endonuclease recognition sites at which such DNA sequencesmay be cut in a determinable fashion without loss of an essentialbiological function of the vector, and into which DNA may be inserted inorder to bring about its replication and cloning. The vector may furthercontain a marker suitable for use in the identification of cellstransformed with the vector.

The techniques and procedures are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. See, e.g., Sambrook, et al. (2001)Molec. Cloning: Lab. Manual [3^(rd) Ed] Cold Spring Harbor LaboratoryPress. Standard techniques may be used for recombinant DNA,oligonucleotide synthesis, and tissue culture, and transformation (e.g.,electroporation, lipofection). Enzymatic reactions and purificationtechniques may be performed according to manufacturer's specificationsor as commonly accomplished in the art or as described herein. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques may be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

Tumor Specific Variants of MUC5AC Comprise a NPC-1 Epitope

The present invention describes peptidomimetics of cancer-specificepitopes on MUC5AC which are specifically bound by NEO-100 seriesantibodies described in International Patent Application No.PCT/US2011/41502 (e.g., NEO-101, NEO-102, NEO-103). The peptidomimeticsdescribed herein, SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ is for L;X² is E or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y;SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO: 2), wherein X¹ is for L; X² is B or D; X¹is Y or W; X⁴ is T or I and X⁵ is Q or Y and SLEPEX¹DWX²FRYX³NY (SEQ IDNO: 3), wherein X¹ is E or D; X² is W or Y; and X³ is T or I; and thepeptidomimetics described in the amino acid sequences of SEQ ID NOs:4-24, mimic epitopes expressed by tumor-specific variants of a MUC5ACantigen, including glycosylation variants. These peptidomimetics may beused in methods for treating and detecting cancer as well as theproduction of tumor-specific antibodies.

Tumor-Specific Variant Form of MUC5AC

A glycosylation variant of MUC5AC is expressed by tumor cells. Thisglycosylation variant may be due to a defect in transferases or otherenzymes involved in glycosylation. MUC5AC isolated from CFPAC-1supernate (pancreatic cancer cell line CFPAC-1) was digested withthermolysin and these fragments (e.g., SEQ ID NOs: 27-33) are bound by aNEO-101 antibody. This lead to the discovery of the NPC-1 epitope, asdisclosed in International Patent Application No. PCT/US2011/41502,which is a tumor-specific glycotype within the tandem repeat region ofMUC5AC. This analysis produced a 15 residue stretch TTSTTSAPTTSTTSAP(SEQ ID NO: 34) that overlaps 100% with the peptides generated from thethermolysin digestion of MUC5AC construct. This region is enriched inProline-Threonine-Serine and may act as a scaffold for aberrantcarbohydrate epitope recognized by a NEO-100 antibody (e.g., NEO-101,NEO-102, NEO-103). This was corroborated by deletion studies of MUC5ACthat suggests the peptide stretch of GCPVTSTPVTAPSTP (SEQ ID NO: 35)binds to a NEO-100 antibody (e.g., NEO-101, NEO-102, NEO-103). Thisregion is believed to act as a scaffold for aberrant glycosylation intumor cells, forming an aberrant glycoprotein pattern that is recognizedby a NEO-100 antibody. The NPC-1 epitope is also sensitive toneuramididase treatment but not to other enzymes (e.g.,β-Glucosaminidase, O-Glycosidase, PNGase F, Neuraminidase (α2→3), β(1→4) galactosidase).

Using peptide phage display, synthetic epitopes that act aspeptidomimetics of the NPC-1 glycotope were identified:SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ is for L; X² is E or D; X³is Y or W; X⁴ is T or I and X⁵ is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO:2), wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is T or I and X⁵is Q or Y and SLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3), wherein X¹ is E or D;X² is W or Y; and X³ is T or I; and the peptidomimetics described in theamino acid sequences of SEQ ID NOs: 4-24. There is no significanthomology between peptide sequences and MUC5AC sequence, which suggestthe peptides comprises NPC-1 epitope peptidomimetics. Such apeptidomimetics are likely to be a glycomimetic of the aberrantglycosylation expressed by tumor cells but not by normal colon orpancreas tissues. This may be useful as a tag in diagnostic assays or acontrol peptide to measure NEO-101 antibody binding. Further, the NPC-1epitope peptidomimetics may be used in diagnostic or therapeutic methodsfor colon, pancreas, stomach, ovarian, lung, breast, or esophaguscancer.

As described in International Patent Application No. PCT/US2011/41502,glycosylation variants of MUC5AC correlate with tumor cells and havecharacterized tumor-specific MUC5AC antigens (e.g., epitopes orantigenic determinants) that may be used in therapeutic and diagnosticmethods (e.g., treatment of cancer involving tumor-specific MUC5ACantigens and the detection of tumor-specific MUC5AC variant antigens.)The immunohistochemistry studies demonstrate that NPC-1 epitope may beuseful as a tissue biomarker of colon, pancreas, stomach, ovarian, lung,breast, or esophagus cancer presence and progression. For example,antibodies targeting the NPC-1 epitope may inhibit tumor progression.Also, NPC-1 epitope levels detected in sera appear to increase as cancerprogresses, thus NPC-1 may be used as a non-invasive diagnostic markerfor colon, pancreas, stomach, ovarian, lung, breast, or esophaguscancer. Thus the NPC-1 epitope peptidomimetic described herein may beused as both a diagnostic and therapeutic target specific for colon,pancreas, stomach, ovarian, lung, breast, or esophagus cancer.

NEO-1 Monoclonal Antibody

As described in International Patent Application No. PCT/US2011/41502,NEO-100 antibodies (e.g., NEO-101, NEO-102, NEO-103) bind to tumor cellsand initiates antibody-dependent cell-mediated cytotoxicity (ADCC) inthis cell and/or inhibits cell proliferation. For example, a NEO-101antibody was produced by means of the hybridoma technique, cloned,chimerized with human constant regions, and also fully humanized. Theinventors surprisingly discovered that the NPC-1 epitope is containedwithin the tandem repeat (TR) regions of the MUC5AC glycoprotein andthat a NEO-100 antibody recognizes an apparently aberrantly glycosylatedform of MUC5AC expressed by tumor cells. This is in contrast with otheranti-MUC5AC antibodies (e.g., 1-13M1, SOMU1, 463M) which predominantlybind near the N-terminus or C-terminus region and not a glycotope in thetandem repeat regions. See Table 1. Further, the NPC-1 epitope issensitive to glycolytic enzymes and thus, suggests that it is aglycotope. Additionally, none of the commercially available antibodiesagainst MUC5AC which were tested by the inventors were found tocross-react with binding by NPC-1. Using these antibodies, the inventorsisolated a peptidomimetic that binds the NEO-101 antibody but does notshare any significant homology with the NPC-1 epitope.

TABLE 1 Compete with NEO-1 Antibody clone Source Binding site antibody?45M1 Abcam Inc. Uncharacterized No H00004586 Abnova Inc. Last 100residues at No carboxyl terminal CLH-2 Millipore Inc. Tandem repeat No2-11M1 Abcam Inc. Amino terminal No 9-13M1 Abcam Inc. Amino terminal No1-13M1 Abcam Inc. TSP-1 Cys-2 region No 2-12M1 Abcam Inc. Carboxylterminal No region No Polyclonal Santa Cruz residues rabbitBiotechnology 1214-1373 (H-160) Inc.

Tumor cell binding activity of NEO-101 was performed by flow cytometryusing colorectal and pancreatic tumor cell lines. As shown in Table 2,the NEO-101 antibody reacted with a sampling of human colorectal andpancreatic tumor cell lines. An isotype control antibody did not reactwith the colorectal and pancreatic tumor cells, demonstrating theantigen-specific reactivity of NEO-101 with these colorectal andpancreatic tumor cell lines. See International Patent Application No.PCT/US2011/41502.

TABLE 2 Flow cytometry: Tumor Cell Binding by NEO-101 % Cells Stained(mfi) Tumor Cell Line Isotype Control NEO-101 LS174T Colorectal 3.85(35) 89.72 (103) Colo-205 Colorectal 2.33 (34) 94.67 (175) SW480Colorectal 3.38 (56) 58.98 (118) CFPAC-1 Pancreatic 1.79 (25) 52.56(59) 

Table 3 shows that 43% of colon cancers and 48% of pancreas cancersstained positively with the NEO-101 antibody. It was observed that onlyone of four normal colon samples showed moderate positivity withNEO-101. Furthermore, in certain instances where normal colon tissuestained positively with NEO-101, the tissue was found to have beensurgically removed from regions adjacent to colon cancer. Consequently,the positively stained “normal” tissues may have already undergonegenotypic changes (“pre-cancerous”) resulting in the expression of theaberrantly glycosylated MUC5AC antigen that could lead to detection ofcarcinoma with NEO-101.

TABLE 3 Immunohistochemistry: Human Tissues With Biotinylated NEO-101Human Tissue staining intensity tissue sample Total (source) NegativeWeak +1 +2 +3 +4 Positive Colon 27/48 5/48 7/48 4/48 5/48 21/48 cancer(56%) (10%) (15%) (8%) (10%) (43%) Normal 3/4 1/4 1/4 colon (75%) (25%)(25%) Pancreas 56/108 17/108 7/108 18/108 10/108 52/108 cancer (52%)(16%) (6%) (17%) (9%) (48%) Normal 3/3 0/3 pancreas (100%) (0%) Uterus32/42 2/42 8/42 10/42 cancer (76%) (5%) (19%) (24%) Normal 12/12 0/12uterus (100%) (0%) Prostate 30/40 5/40 5/40 10/40 cancer (75%) (12%)(12%) (25%) Normal 4/4 0/4 prostate (100%) (0%)

Staining with a human IgG1 isotype control antibody showed no reactivityagainst the same tissues. Immunohistochemical studies demonstrateNEO-101 tissue staining in pancreatic adenocarcinoma tissue, and lack ofstaining in normal pancreas tissue.

In summary, antibody-staining results with NEO-101 demonstrated specificimmunoreactivity with cancer tissues from colon and pancreas patients,whereas only weak binding, if at all, was observed in normal pancreas orcolon tissues. Furthermore, no cross-reactivity was observed in othernormal human tissues stained, indicating a strong positive correlationof the NEO-101 binding to colon and pancreas cancer tissues. Thus, theNPC-1 epitope is expressed by colon and pancreatic tumor cells but notnormal colon or pancreatic tumor cells. Therefore, the NPC-1 epitope maybe used as a tumor-specific marker or a therapeutic target for colon andpancreatic cancer. Further, peptidomimetics of the NPC-1 epitope (e.g.,the polypeptide of SEQ ID NO: 1-24) as described herein may be used inthe detection and treatment of cancer (e.g., colon, pancreas, breast,lung, ovarian, stomach, esophageal).

NPC-1 Epitope Peptidomimetics

The invention provides NPC-1 epitope peptidomimetics. The inventorssurprisingly discovered that MUC5AC comprises at least one NPC-1epitope. Exemplary polypeptides comprising at least one NPC-1 epitopeare provided in GCPVTSTPVTAPSTP (SEQ ID NO: 35). The peptidomimetics ofthe NPC-1 epitope may comprise the following sequences:SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO: 1), wherein X¹ is for L; X² is E or D; X³is Y or W; X⁴ is T or I and X⁵ is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO:2), wherein X¹ is for L; X² is E or D; X³ is Y or W; X¹ is T or I and X⁵is Q or Y and SLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3), wherein X¹ is E or D;X² is W or Y; and X³ is T or I; FPEDYFRYTNQK (SEQ ID NO: 4);SLPDDWFRYINY (SEQ ID NO: 5); and the peptidomimetics described in theamino acid sequences of SEQ ID NOs: 6-24.

Nucleic acids encoding polypeptides comprising at least one NPC-1epitope peptidomimetic may be modified using standard molecularbiological techniques that result in variants polypeptides comprising atleast one NPC-1 epitope including but not limited to deletions,additions and substitutions in the amino acid sequence, that retain thespecific antigenicity of the NPC-1 epitope (e.g., the NPC-1 epitope isbound by the NEO-1 antibody). Additionally, variant polypeptidescomprising at least one NPC-1 epitope may also retain the antigenicityof a NPC-1 epitope (e.g., raise a specific immune response against theNPC-1 epitopes, respectively, upon immunization in a subject). The NPC-1epitope peptidomimetics may be formulated with a pharmaceutical carrierto manufacture an antigen composition useful as a “cancer vaccine”(e.g., a pharmaceutical composition that elicits a specific immuneresponse against the NPC-1 epitope, that produces anti-tumor antibodiesafter immunization in a subject). For example, a cancer vaccine maycomprise a composition comprising a polypeptide comprising an amino acidsequence of any one of SEQ ID NOs: 1-24, or combinations thereof. Acancer vaccine may comprise a composition comprising a polypeptidecomprising an amino acid sequence of any one of SEQ ID NO: 4 or 5 and anadjuvant or pharmaceutical carrier. Also, a cancer vaccine may comprisea composition comprising a polypeptide comprising an amino acid sequenceof any one of SEQ ID NO: 4 or 5 conjugate to a carrier such as KLH. TheNPC-1 epitope peptidomimetic may be a small peptide. Further, thepeptide structure of the peptidomimetic may have an altered chemicalstructure is designed to improve stability or biological activity. Suchmodifications include altered backbones and the incorporation ofnonnatural amino acids.

Polypeptide Derivatives and Analogs

It will be appreciated that the NPC-1 epitope peptidomimetics describedherein may be polypeptides, degradation products, synthetic peptides, orrecombinant peptides as well as peptidomimetics, synthetic peptides,peptoids, and semipeptoids (e.g., peptide analogs, which may have, forexample, modifications rendering the peptides more stable while in abody or more capable of penetrating into cells.) Modifications of theNPC-1 epitope peptidomimetics described herein include, but are notlimited to N-terminus modification, C-terminus modification, peptidebond modification (e.g., CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂,S═C—NH, CH═CH or CF═CH), backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art. Martin (2010) Quantitative Drug Design: A CriticalIntroduction [2^(nd) Ed.] CRC Press.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH₂—), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bysynthetic non-natural acid such as phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of phenylalanine, halogenatedderivatives of phenylalanine or o-methyl-tyrosine. In addition to theabove, the polypeptides of the present invention may also include one ormore modified amino acids or one or more non-amino acid monomers (e.g.fatty acids, complex carbohydrates), for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Since the polypeptides of the present invention are preferably utilizedin therapeutics which requires the peptides to be in soluble form, thepolypeptides of the present invention may comprise one or morenon-natural or natural polar amino acids, including but not limited toserine and threonine which are capable of increasing peptide solubilitydue to their hydroxyl-containing side chain.

The NPC-1 epitope peptidomimetics of the present invention may be in alinear form, although it will be appreciated that circular forms mayalso be utilized.

The NPC-1 epitope peptidomimetics described herein may be purified fromcells that have been altered to express it (e.g., recombinant). DNAsequences encoding the NPC-1 epitope peptidomimetics may be insertedinto an expression vector and then transformed (or transfected) in anappropriate host cell and/or expressed in a transgenic animal. The NPC-1epitope peptidomimetics so expressed may then be isolated by methodsknown in the art. See, e.g., Maniatis, et al. (2001) Molecular Cloning:A Laboratory Manual [3^(rd) Ed.] Cold Spring Harbor Laboratory Press.

The NPC-1 epitope peptidomimetics of the present invention may bebiochemically synthesized such as by using standard solid phasetechniques. These methods include exclusive solid phase synthesis,partial solid phase synthesis methods, fragment condensation, classicalsolution synthesis. Solid phase peptide synthesis procedures are wellknown in the art and further described by Stewart (1984) Solid PhasePeptide Syntheses [2^(nd) Ed.] Pierce Chemical Company and Benoiton(2005) Chemistry of Peptide Synthesis CRC Press. Synthetic peptides maybe purified by preparative high performance liquid chromatography andthe composition of which may be confirmed via amino acid sequencing. SeeCreighton (1992) [2^(nd) Ed.] Proteins, Structures and MolecularPrinciples W.H. Freeman and Company; Aguilar (2004) [Ed.] HPLC ofPeptides and Proteins: Methods and Protocols Humana Press; Simpson(2002) Protein Sequencing Protocols [2^(nd) Ed.] Humana Press.

In cases where large amounts of the NPC-1 epitope peptidomimetics of thepresent invention are desired, the NPC-1 epitope peptidomimetics of thepresent invention may be generated using recombinant techniques such asdescribed by Invitrogen (2002) “Guide to Baculovirus Expression VectorSystems (BEVs) and Insect Culture Techniques” Instruction Manual;Hatti-Kaul and Mattiasson (2003) [Eds] Isolation and Purification ofProteins: Ahmed (2004) Principles and Reactions of Protein Extraction.Purification and Characterization CRC Press. Further recombinanttechniques such as described by, for example, Bitter, et al. (1987)Methods in Enzymol. 153: 516-544, Studier, et al. (1990) Methods inEnzymol. 185: 60-89, Brisson, et al. (1984) Nature 310: 511-514,Takamatsu, et al. (1987) EMBO J. 6: 307-311, Coruzzi, et al. (1984) EMBOJ. 3: 1671-1680 and Brogli, et al. (1984) Science 224: 838-843, Gurley,et al. (1986) Mol. Cell. Biol. 6: 559-565 and Weissbach & Weissbach(1988) Methods for Plant Molecular Biology. Academic Press, NY, SectionVIII, pages 421-463.

Polypeptide Sequence Variants

For any NPC-1 epitope peptidomimetic sequence described herein, furthercharacterization or optimization may be achieved by systematicallyeither adding or removing amino acid residues to generate longer orshorter peptides, and testing those and sequences generated by walking awindow of the longer or shorter size up or down the antigen from thatpoint. Coupling this approach to generating new candidate targets withtesting for effectiveness of antigenic molecules based on thosesequences in an immunogenicity assay, as known in the art or asdescribed herein, may lead to further manipulation of the antigen.Further still, such optimized sequences may be adjusted by, e.g., theaddition, deletions, or other mutations as known in the art and/ordiscussed herein to further optimize the NPC-1 epitope peptidomimetic(e.g., increasing serum stability or circulating half-life, increasingthermal stability, enhancing delivery, enhance immunogenicity,increasing solubility, targeting to a particular in vivo location orcell type).

The NPC-1 epitope peptidomimetics described herein may compriseconservative substitution mutations, (i.e., the substitution of one ormore amino acids by similar amino acids). For example, conservativesubstitution refers to the substitution of an amino acid with anotherwithin the same general class, e.g., one acidic amino acid with anotheracidic amino acid, one basic amino acid with another basic amino acid,or one neutral amino acid by another neutral amino acid.

NPC-1 epitope peptidomimetic sequences may have at least about 60, 65,70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% sequence homology to any at least one of thepolypeptide sequences of SEQ ID NOs: 1-24. Further, the variant NPC-1epitope peptidomimetics described herein may retain the antigeniticityof the sequence from which they were derived. More preferably, theinvention contemplates polypeptide sequences having at least about 95%sequence homology, even more preferably at least about 98% sequencehomology, and still more preferably at least about 99% sequence homologyto any one or more of the polypeptide sequences of NPC-1 epitopepeptidomimetic sequences set forth in SEQ ID NOs: 1-24. Methods fordetermining homology between amino acid sequences, as well as nucleicacid sequences, are well known to those of ordinary skill in the art.See, e.g., Nedelkov & Nelson (2006) New and Emerging ProteomicTechniques Humana Press.

Thus, a NPC-1 epitope peptidomimetic may have at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence homology with a polypeptidesequence of SEQ ID NOs: 1-24. For example, a NPC-1 epitopepeptidomimetic may have at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence homology with SEQ ID NOs: 1-24. The variant NPC-1 epitopepeptidomimetics described herein may retain the antigeniticity of thesequence from which they were derived (e.g., a variant NPC-1 epitopepeptidomimetic with at least about 80% homology to the amino acidsequence of SEQ ID NO: 5 may have the same antigenicity as a polypeptidewith the amino acid sequence of SEQ ID NO: 5).

The term homology, or identity, is understood as meaning the number ofagreeing amino acids (identity) with other proteins, expressed inpercent. The identity is preferably determined by comparing a givensequence with other proteins with the aid of computer programs. Ifsequences which are compared with each other are different in length,the identity is to be determined in such a way that the number of aminoacids which the short sequence shares with the longer sequencedetermines the percentage identity. The identity can be determinedroutinely by means of known computer programs which are publiclyavailable such as, for example, ClustalW. Thompson, et al. (1994)Nucleic Acids Research 22: 4673-4680. ClustalW is publicly availablefrom the European Molecular Biology Laboratory and may be downloadedfrom various internet pages, inter alia the IGBMC (Institut de Génétiqueet de Biologie Moléculaire et Cellulaire) and the EBI and all mirroredEBI internet pages (European Bioinformatics Institute). If the ClustalWcomputer program Version 1.8 is used to determine the identity between,for example, the reference protein of the present application and otherproteins, the following parameters are to be set: KTUPLE=1, TOPDIAG=5,WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40,MATRIX=GONNET, ENDGAPS(OFP), NOPGAP, NOHGAP. See also EuropeanBioinformatics Institute (EBI) toolbox available on-line and Smith(2002) Protein Sequencing Protocols [2^(nd) Ed.] Humana Press.

One possibility of finding similar sequences is to carry out sequencedatabase researches. Here, one or more sequences may be entered as whatis known as a query. This query sequence is then compared with sequencespresent in the selected databases using statistical computer programs.Such database queries (blast searches) are known to the skilled workerand may be carried out at different suppliers. If, for example, such adatabase query is carried out at the NCBI (National Center forBiotechnology Information), the standard settings for the respectivecomparison query should be used. For protein sequence comparisons(blastp), these settings are: Limit entrez=not activated; Filter=lowcomplexity activated; Expect value=10; word size=3; Matrix=BLOSUM62; Gapcosts: Existence=11, Extension=1. The result of such a query is, amongother parameters, the degree of identity between the query sequence andthe similar sequences found in the databases. NPC-1 epitopepeptidomimetics include functional fragments of said peptidomimetics. A“functional fragment” of said polypeptide includes a fragment of thegene or cDNA encoding said NPC-1 epitope, which fragment is capable ofeliciting an immune response (e.g., humoral or cellular immuneresponse.) Thus, for example, fragments of the NPC-1 epitope accordingto the invention which correspond to amino acid residues that contributeto the immunogenicity of the antigen and which fragments may serve tofunction as antigens to elicit an immune response (e.g., humoral orcellular immune response.) This aspect of the invention also includesdifferentially spliced isoforms and transcriptional starts of thepolypeptides according to the invention. The polypeptides according tothe invention also may comprise fragments, derivatives and allelicvariants of the NPC-1 epitope peptidomimetics. Methods and materials formaking fragments of NPC-1 epitope peptidomimetics are well known in theart. See, e.g., Maniatis, et al. (2001) Molecular Cloning: A LaboratoryManual [3^(rd) Ed.] Cold Spring Harbor Laboratory Press.

Variant NPC-1 epitope peptidomimetics may retain their antigenicspecificity to bind their respective antibodies (e.g., a variant NPC-1epitope peptidomimetic binds NEO-101 antibody.) Fully antigenic variantsmay contain only conservative variations or variations in non-criticalresidues or in non-critical regions. Antigenic variants may also containsubstitution of similar amino acids that result in no change or aninsignificant change in antigenicity. Alternatively, such substitutionsmay positively or negatively affect antigenicity to some degree.Non-antigenic variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region of an epitope. Molecular biology andbiochemistry techniques for modifying NPC-1 epitope peptidomimeticswhile preserving specific antigenicity of the polypeptides for theirrespective antibodies are well known in the art. See, e.g., Ho, et al.(1989) Gene 77(1): 51-59; Landt, et al. (1990) Gene 96(1): 125-128; Hopp& Woods (1991) Proc. Natl. Acad. Sci. USA 78(6): 3824-3828; Kolaskar &Tongaonkar (1990) FEBS Letters 276(1-2): 172-174; and Welling, et al.(1985) FEBS Letters 188(2): 215-218

Amino acids that are essential for function may be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis. Cunningham, et al. (1989) Si 244: 1081-85. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as epitope binding or in vitro ADCC activity. Sites thatare critical for ligand-receptor binding may also be determined bystructural analysis such as crystallography, nuclear magnetic resonance,or photoaffinity labeling. Smith, et al. (1992) J. Mol. Biol. 224:899-904; de Vos, et al. (1992) Sci. 255: 306-12.

For example, one class of substitutions is conserved amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid in a NPC-1 epitope peptidomimetic with another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Arg,replacements among the aromatic residues Phe, Tyr. Guidance concerningwhich amino acid changes are likely to be phenotypically silent is foundin, for example, Bowie, et al. (1990) Sci. 247: 1306-10. Hence, one ofordinary skill in the art appreciates that the inventors possess peptidevariants without delineation of all the specific variants. As to aminoacid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. See, e.g., Creighton (1992)Proteins: Structures and Molecular Properties [2^(nd) Ed.] W.H. Freeman.

Moreover, the NPC-1 epitope peptidomimetics may contain amino acidsother than the twenty “naturally occurring” amino acids. Further, manyamino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, g-carboxylation, glycosylation, GPI anchorformation, hydroxylation, iodination, methylation, myristoylation,oxidation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination. SeeCreighton (1992) Proteins: Structure and Molecular Properties [2^(nd)Ed.] and Lundblad (1995) Techniques in Protein Modification [1^(st) Ed.]Many detailed reviews are available on this subject. See, e.g., Wold(1983) Posttranslational Covalent Modification of Proteins Acad. Press,NY; Seifter, et al. (1990) Meth. Enzymol. 182: 626-46; and Rattan, etal. (1992) Ann. NY Acad. Sci. 663: 48-62.

Fusion Proteins

Fusions comprising the NPC-1 epitope peptidomimetics are also within thescope of the present invention. For example, the fusion proteins maycomprise a NPC-1 epitope peptidomimetic sequence fused to the C-terminusof the GST sequences. Such fusion proteins may facilitate thepurification of the recombinant NPC-1 epitope peptidomimetics.Alternatively, NPC-1 epitope peptidomimetics may be fused with a proteinthat binds B-cell follicles, thus initiating both a humoral immuneresponse and activation of T cells. Berney, et al. (1999) J. Exp. Med.190: 851-60. Alternatively, for example, the NPC-1 epitopepeptidomimetics may be genetically coupled with and anti-dendritic cellantibody to deliver the antigen to the immune system and stimulate acellular immune response. He, et al. (2004) Clin. Cancer Res. 10:1920-27. A chimeric or fusion protein of the invention may be producedby standard recombinant DNA techniques. For example, DNA fragmentscoding for the different polypeptide sequences are ligated togetherin-frame in accordance with conventional techniques, e.g., by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. The fusion gene may be synthesized byconventional techniques including automated DNA synthesizers.

Fusion proteins may include C-terminal or N-terminal translocationsequences. Further, fusion proteins comprising NPC-1 epitopepeptidomimetics may comprise additional elements, e.g., for proteindetection, purification, or other applications. Detection andpurification facilitating domains including but not limited to metalchelating peptides such as polyhistidine tracts, histidine-tryptophanmodules, or other domains that allow purification on immobilized metals;maltose binding protein; protein A domains that allow purification onimmobilized immunoglobulin; or the domain utilized in the FLAGextension/affinity purification system (Immunex Corp, Seattle Wash.)

A fusion protein comprising a NPC-1 epitope peptidomimetic may beprepared from a protein of the invention by fusion with a portion of animmunoglobulin comprising a constant region of an immunoglobulin. Morepreferably, the portion of the immunoglobulin comprises a heavy chainconstant region which is optionally and more preferably a human heavychain constant region. The heavy chain constant region is mostpreferably an IgG heavy chain constant region, and optionally and mostpreferably is an Fc chain, most preferably an IgG Fc fragment thatcomprises CH2 and CH3 domains. Although any IgG subtype may optionallybe used, the IgG1 subtype is preferred. The Fc chain may optionally be aknown or “wild type” Fc chain, or alternatively may be mutated. See,e.g., U.S. Patent Application Publication No. 2006/0034852. The term “Fcchain” also optionally comprises any type of Fc fragment. Several of thespecific amino acid residues that are involved in antibody constantregion-mediated activity in the IgG subclass have been identified.Inclusion, substitution or exclusion of these specific amino acidstherefore allows for inclusion or exclusion of specific immunoglobulinconstant region-mediated activity. Furthermore, specific changes mayresult in aglycosylation for example and/or other desired changes to theFc chain. At least some changes may optionally be made to block afunction of Fc which is considered to be undesirable, such as anundesirable immune system effect. See McCafferty, et al. (2002) AntibodyEngineering: A Practical Aooroach (Eds.) Oxford University Press.

The inclusion of a cleavable linker sequences such as Factor Xa (see,e.g., Ottavi, (1998) Biochimie 80: 289-93), subtilisin proteaserecognition motif (see, e.g., Polyak (1997) Protein Eng. 10: 615-19);enterokinase (Invitrogen, San Diego, Calif.), between the translocationdomain (for efficient plasma membrane expression) and the rest of thenewly translated polypeptide may be useful to facilitate purification.For example, one construct can include a polypeptide encoding a nucleicacid sequence linked to six histidine residues followed by athioredoxin, an enterokinase cleavage site (see, e.g., Williams (1995)Biochmistry 34: 1787-97), and an C-terminal translocation domain. Thehistidine residues facilitate detection and purification while theenterokinase cleavage site provides a means for purifying the desiredprotein(s) from the remainder of the fusion protein. Technologypertaining to vectors encoding fusion proteins and application of fusionproteins are well described in the scientific and patent literature.See, e.g., Kroll (1993) DNA Cell. Biol. 12: 441-53.

Conjugates

The NPC-1 epitope peptidomimetics may be conjugated to other moieties.Such conjugates are often used in the preparation of vaccines. The NPC-1epitope peptidomimetic may be conjugated to a carbohydrate carrier(e.g., mannose, fucose, glucose, GlcNAs, maltose), which is recognizedby the mannose receptor present on dendritic cells and macrophages. Theensuing binding, aggregation, and receptor-mediated endocytosis andphagocytosis functions provide enhanced innate and adaptive immunity.See Mahnke, et al. (2000) J. Cell Biol. 151: 673-84; Dong, et al. (1999)J. Immonol. 163: 5427-34. Other moieties suitable for conjugationinclude carriers which elicit an immune response includes but notlimited to Keyhole Limpit Hemocyannin (KLH), ovalbumin, bovine serumalbumin (BSA), diphtheria toxoid, cholera toxoid, Pseudomonas exoproteinA, and microbial outer membrane proteins (OMPS). Further, NPC-1 epitopepeptidomimetic conjugates may be used to raise antibodies including butnot limited to monoclonal antibodies that bind the NPC-1 epitopepeptidomimetic and thus, selectively bind MUC5AC in tumor cells but notnormal cells.

Polypeptide Isolation

The present invention also provides methods for isolation of the NPC-1epitope peptidomimetics. For example, relevant cell lines or tumorsamples may be obtained from a cancer patient. After homogenization andsolubilization in a detergent, the antigen is chromatographicallypurified. Size-exclusion or affinity chromatography may be used forthis, and may be used in conjunction with NEO-101 antibody binding. SeeInternational Patent Application No. PCT/US2011/041502 for a descriptionof the NEO-100 antibodies including the NEO-101 antibody. For example,NEO-101 antibody may be immobilized on a solid support (e.g., coupled toresins, magnetic beads) for simple antigen adsorption, washing, andelution from the solid support. The eluted protein is then studiedfurther for antigen presence, characterization, and identification. SeeWalker (2002) Protein Protocols Handbook [2^(nd) Ed.] Humana Press andCultur (2003) [Ed.] Protein Purification Protocols Humana Press.

The antigen isolated in this way may be used for preparing apharmaceutical using the conventional pharmaceutical excipient andcarrier substance. For example, in-vivo administration of the purifiedantigen in a physiological NaCl solution.

Additionally, the NPC-1 epitope peptidomimetics according to theinvention may serve as an antigen in the identification of activities aspart of a high-throughput screening. High-throughput screening methodsare known to persons skilled in the art. Wells (2002) High ThroughoutBioanalytical Sample Preparation Elsevier Health Sciences.

Antibodies which Bind NPC-1 Epitope Peptidomimetic

The present invention also provides antibodies that bind the NPC-1epitope peptidomimetic including but not limited monoclonal andhumanized monoclonal antibodies (e.g., NEO-101 antibody as described inInternational Patent Application No. PCT/US2011/041502). Such antibodiesalso selectively bind aberrant MUC5AC in tumor cells but not normalcells (e.g., healthy cells). The NPC-1 epitope peptidomimetics bindingantibodies may be admixed in compositions with pharmaceutical carriersand antibodies (e.g., NEO-201 and/or NEO-301 monoclonal antibodies).Exemplary NPC-1 binding antibodies (e.g., NEO-100 antibodies) areprovided in Table 4.

TABLE 4 NEO-100 Series Antibodies which selectively bind a NPC-1epitope. Antibody Aliases Antigen Exemplary SEQ ID NOs Description NPC-1NPC-1 Murine hybridoma that expresses NPC-1 IgG1 (ATCC) NEO-101 NPC-1C,NPC-1 Light Chain (SEQ ID NOs: 57, 58) Chimeric NEO-101 antibody,ensituximab LC CDRs (SEQ ID NOs: 59-61) engineered in CHO-DG44 HeavyChain (SEQ ID NOs: 62, 63) production cell clone 4B7; targets a HC CDRs(SEQ ID NOs: 64-66) variant of MUC5AC NEO-102 NPC-1 Light Chain (SEQ IDNOs: 67, 68) Chimeric NEO-101 antibody, LC CDRs (SEQ ID NOs: 69-71)engineered in CHO-M production Heavy Chain (SEQ ID NOs: 72, 73) cells,contains 2 amino acid changes HC CDRs (SEQ ID NOs: 74-76) in HC constantdomain* NEO-103 NPC-1 Light Chain (SEQ ID NOs: 77, 78) Humanized NEO-101antibody Heavy Chain (SEQ ID NOs: 79, 80)

Antibodies may comprise of two identical light polypeptide chains ofmolecular weight approximately 23,000 daltons (“light chain”), and twoidentical heavy chains of molecular weight 53,000-70,000 (“heavychain”). See Edelman (1971) Ann, NY. Acad. Sci. 190: 5. The four chainsare joined by disulfide bonds in a “Y” configuration wherein the lightchains bracket the heavy chains starting at the mouth of the “Y”configuration. The “branch” portion of the “Y” configuration isdesignated the F_(ab) region; the stem portion of the “Y” configurationis designated the Fc region. The amino acid sequence orientation runsfrom the N-terminal end at the top of the “Y” configuration to theC-terminal end at the bottom of each chain. The N-terminal end possessesthe variable region having specificity for the antigen that elicited it,and is about 100 amino acids in length, there being slight variationsbetween light and heavy chain and from antibody to antibody.

The variable region is linked in each chain to a constant region thatextends the remaining length of the chain and that within a particularclass of antibody does not vary with the specificity of the antibody(i.e., the antigen eliciting it). There are five known major classes ofconstant regions that determine the class of the immunoglobulin molecule(e.g., IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and εheavy chain constant regions). The constant region or class determinessubsequent effector function of the antibody, including activation ofcomplement (Kabat (1976) Structural Concepts in Immunology andImmunochemistry [2^(nd) Ed.] pages 413-436; Holt, Rinehart, Winston) andother cellular responses (Andrews, et al. (1980) Clinical Immunobiology1-18; Kohl, et al. (1983) Immunology 48: 187) while the variable regiondetermines the antigen with which it will react. Light chains areclassified as either κ (kappa) or λ (lambda). Each heavy chain class maybe prepared with either kappa or lambda light chain. The light and heavychains are covalently bonded to each other, and the “tail” portions ofthe two heavy chains are bonded to each other by covalent disulfidelinkages when the immunoglobulins are generated either by hybridomas orby B cells.

Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to seminal basic protein fromspecific species such as rat, mouse, or human can be selected to obtainonly those polyclonal antibodies that are specifically immunoreactivewith seminal basic protein and not with other proteins, except forpolymorphic variants and alleles of seminal basic protein. Thisselection may be achieved by subtracting out antibodies that cross-reactwith seminal basic protein molecules from other species. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow & Lane (1998) USINGANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than about 10 to 100 times background.

Polyclonal Antibody

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen.Polyclonal antibodies which selectively bind the NPC-1 epitopepeptidomimetic may be made by methods well-known in the art. See, e.g.,Howard & Kaser (2007) Making and Using Antibodies: A Practical HandbookCRC Press.

Monoclonal Antibody

A monoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. Monoclonal antibodies may be obtained bymethods known to those skilled in the art. See, e.g. Kohler and Milstein(1975) Nature 256: 495-497; U.S. Pat. No. 4,376,110; Ausubel, et al.[Eds.] (2011) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. Greene PublishingAssoc. and Wiley Interscience, NY.; and Harlow & Lane (1998) USINGANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory; Colligan,et al. (2005) [Eds.] Current Protocols in Immunology Greene PublishingAssoc. and Wiley Interscience, NY. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclassthereof. A hybridoma producing an antibody of the present invention maybe cultivated in vitro, in situ, or in vivo. Examples of monoclonalantibodies that bind a NPC-1 epitope peptidomimetic include but are notlimited to an NEO-101 antibody which selectively binds the NPC-1 epitope(e.g., exemplary light chain are depicted in SEQ ID NO: 57, 58 with CDRsdepicted in SEQ ID NO: 59-61 and heavy chain are depicted in SEQ ID NO:62, 63 with CDRs depicted in SEQ ID NO: 64-66, exemplary light chain aredepicted in SEQ ID NO: 67, 68 with CDRs depicted in SEQ ID NO: 69-71 andheavy chain are depicted in SEQ ID NO: 72, 73 with CDRs depicted in SEQID NO: 74-76, and exemplary light chain are depicted in SEQ ID NO: 77,78 and heavy chain are depicted in SEQ ID NO: 79, 80).

Chimeric Antibody

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having variableregion derived from a murine antibody and a human immunoglobulinconstant region, which are primarily used to reduce immunogenicity inapplication and to increase yields in production, for example, wheremurine monoclonal antibodies have higher yields from hybridomas buthigher immunogenicity in humans, such that human murine chimericmonoclonal antibodies are used. Chimeric antibodies and methods fortheir production are known in the art. See Cabilly, et al. (1984) Proc.Natl. Acad. Sci. USA 81: 3273-3277; Morrison, et al. (1994) Proc. Natl.Acad. Sci. USA 81: 6851-6855, Boulianne, et al. (1984) Nature 312:643-646; Neuberger, et al. (1985) Nature 314: 268-270; European PatentApplication 173494 (1986); WO 86/01533 (1986); European PatentApplication 184187 (1986); Sahagan, et al. (1986) J. Immunol. 137:1066-1074; Liu, et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443;Sun, et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Better, etal. (1988) Science 240: 1041-1043; and Harlow & Lane (1998) USINGANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory; U.S. Pat.No. 5,624,659. Exemplary chimeric antibodies include but are not limitedto NEO-101 (NEO-101) which selectively binds NPC-1 epitope (e.g.,exemplary light chain are depicted in SEQ ID NOs: 52, 58 with CDRSdepicted in SEQ ID NOs: 59-61 and heavy chain depicted in SEQ ID NOs:62, 63 with CDRs depicted in SEQ ID NOs: 64-66); NEO-102 whichselectively binds NPC-1 epitope (e.g., exemplary light chain aredepicted in SEQ ID NOs: 67, 68 with CDRS depicted in SEQ ID NOs: 69-71and heavy chain depicted in SEQ ID NOs: 72, 73 with CDRs depicted in SEQID NOs: 74-76).

Humanized Antibody

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only thecomplementarity-determining regions of the animal-derived antibody. Thismay be accomplished by examining the sequence of the hyper-variableloops of the variable regions of the monoclonal antibody, and fittingthem to the structure of the human antibody chains. See, e.g., U.S. Pat.No. 6,187,287. Likewise, other methods of producing humanized antibodiesare now well known in the art. See, e.g., U.S. Pat. Nos. 5,225,539;5,530,101; 5,585,089; 5,693,762; 6,054,297; 6,180,370; 6,407,213;6,548,640; 6,632,927; and U.S. Pat. No. 6,639,055; Jones, et al. (1986)Nature 321: 522-525; Reichmann, et al. (1988) Nature 332: 323-327;Verhoeyen, et al. (1988) Science 239: 1534-36; and Zhiqiang An (2009)[Ed.] Therapeutic Monoclonal Antibodies: From Bench to Clinic John Wiley& Sons, Inc. Examples of humanized antibodies include but are notlimited to NEO-103 which selectively binds the NPC-1 epitope (e.g.,exemplary light chain are depicted in SEQ ID NO: 77, 78 and heavy chainare depicted in SEQ ID NO: 79, 80).

Antibody Fragments

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment,” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Pv” immunoglobulinsfor use in the present invention may be produced by synthesizing a fusedvariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificitics. Antigen-binding fragments ofimmunoglobulins include but are not limited to SMIPs (small moleculeimmunopharmaceuticals), camelbodies, nanobodies, and IgNAR.

Anti-Idiotypic Antibody

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody may be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the antibody with the antibody to which an anti-Id is being prepared.The immunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See e.g., U.S. Pat.No. 4,699,880. The anti-Id antibody may also be used as an “immunogen”to induce an immune response in yet another animal, producing aso-called anti-anti-Id antibody. The anti-anti-Id may be epitopicallyidentical to the original antibody which induced the anti-Id. Thus, byusing antibodies to the idiotypic determinants of an antibody it ispossible to identify other clones expressing antibodies of identicalspecificity. An exemplary anti-idiotypic antibody is 4B6, whichselectively binds the NEO-101 antibody, both of which are described inInternational Patent Application No. PCT/US2011/041502. Thisanti-idiotypic antibody specific for NEO-1 antibody. In one embodiment,the light chain of said antibody may be encoded by the nucleic acidsequence of SEQ ID NO: 81. In one embodiment, the light chain of saidantibody may comprise the amino acid sequence of SEQ ID NO: 82. In oneembodiment, the light chain of said antibody may comprise CDRscomprising the amino acid sequence of SEQ ID NO: 83 and 84 and thepeptide sequence Trp-Ala-Ser. In one embodiment, the heavy chain of saidantibody may be encoded by the nucleic acid sequence of SEQ ID NO: 85.In one embodiment, the heavy chain of said antibody may comprise theamino acid sequence of SEQ ID NO: 86. In one embodiment, the heavy chainof said antibody may comprise CDRs comprising the amino acid sequence ofSEQ ID NO: 87, 88, and 89.

Engineered and Modified Antibodies

An antibody of the invention further may be prepared using an antibodyhaving one or more of the VH and/or VL sequences derived from anantibody starting material to engineer a modified antibody, whichmodified antibody may have altered properties from the startingantibody. An antibody may be engineered by modifying one or moreresidues within one or both variable regions (i.e., VH and/or VL), forexample within one or more CDR regions and/or within one or moreframework regions. Additionally or alternatively, an antibody may beengineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that may be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties. See, e.g., Riechmann, et al. (1998) Nature 332:323-327; Jones, et al. (1986) Nature 321: 522-525; Queen, et al. (1989)Proc. Natl. Acad. U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539;5,530,101; 5,585,089; 5,693,762; and 6,180,370.

Suitable framework sequences may be obtained from public DNA databasesor published references that include germline antibody gene sequences.For example, germline DNA sequences for human heavy and light chainvariable region genes may be found in the “VBase” human germlinesequence database (available on the Internet), as well as in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest. FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242; Tomlinson, et al. (1992) “The Repertoire of Human GermlineVH Sequences Reveals about Fifty Groups of VH Segments with DifferentHypervariable Loops” J. Mol. Biol. 227: 776-798; and Cox, et al. (1994)Eur. J Immunol. 24: 827-836.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR 1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis may be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, may beevaluated in appropriate in vitro or in vivo assays. Preferablyconservative modifications (as discussed herein) may be introduced. Themutations may be amino acid substitutions, additions or deletions, butare preferably substitutions. Moreover, typically no more than one, two,three, four or five residues within a CDR region are altered.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation may contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues may be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties may be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Suchembodiments are described further below. The numbering of residues inthe Fc region is that of the EU index of Kabat.

The hinge region of CH1 may be modified such that the number of cysteineresidues in the hinge region is altered, e.g., increased or decreased.See U.S. Pat. No. 5,677,425. The number of cysteine residues in thehinge region of CH1 may be altered to, for example, facilitate assemblyof the light and heavy chains or to increase or decrease the stabilityof the antibody.

The Fc hinge region of an antibody may be mutated to decrease thebiological half life of the antibody. More specifically, one or moreamino acid mutations may be introduced into the CH2-CH3 domain interfaceregion of the Fc-hinge fragment such that the antibody has impairedStaphylococcyl protein A (SpA) binding relative to native Fc-hingedomain SpA binding. See, e.g., U.S. Pat. No. 6,165,745.

The antibody may be modified to increase its biological half life.Various approaches are possible. For example, one or more of thefollowing mutations may be introduced: T252L, T254S, T256F. See U.S.Pat. No. 6,277,375. Alternatively, to increase the biological half life,the antibody may be altered within the CH1 or CL region to contain asalvage receptor binding epitope taken from two loops of a CH2 domain ofan Fc region of an IgG. See U.S. Pat. Nos. 5,869,046 and 6,121,022.

The Fc region may be altered by replacing at least one amino acidresidue with a different amino acid residue to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 may be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity may be altered may be, for example, an Fc receptor or theC1 component of complement. See U.S. Pat. Nos. 5,624,821 and 5,648,260.

The Fc region may be modified to increase the ability of the antibody tomediate antibody dependent cellular cytotoxicity (ADCC) and/or toincrease the affinity of the antibody for an Fcy receptor by modifyingone or more amino acids at the following positions: 238, 239, 248, 249,252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280,283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334,335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416,419, 430, 434, 435, 437, 438 or 439. See WO 00/42072. Moreover, thebinding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding. See Shields, et al.(2001) J. Biol. Chem. 276: 6591-6604. Specific mutations at positions256, 290, 298, 333, 334 and 339 are shown to improve binding to FcγRIII.Additionally, the following combination mutants are shown to improveFcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A andS298A/E333A/K334A.

The glycosylation of an antibody may be modified. For example, anaglycoslated antibody may be made (i.e., the antibody lacksglycosylation). Glycosylation may be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications may be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions may be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos.5,714,350 and 6,350,861.

Additionally or alternatively, an antibody may be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications may be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and may be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. See U.S. Patent Application Publication No.2004/0110704 and Yamane-Ohnuki, et al. (2004) Biotechnol Bioeng. 87:614-22; EP 1176195 (2002); WO 2003/035835; Shields, et al. (2002) J.Biol. Chem. 277: 26733-26740; WO 99/54342; Umana, et al. (1999) Nat.Biotech. 17: 176-180; and Tarentino, et al. (1975) Biochem. 14: 5516-23.

An antibody may be pegylated to, for example, increase the biological(e.g., serum) half life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer).

The invention also provides variants and equivalents that aresubstantially homologous to the antibodies, antibody fragments,diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variableregions and CDRs set forth herein. These may contain, e.g., conservativesubstitution mutations, (i.e., the substitution of one or more aminoacids by similar amino acids). For example, conservative substitutionrefers to the substitution of an amino acid with another within the samegeneral class, e.g., one acidic amino acid with another acidic aminoacid, one basic amino acid with another basic amino acid, or one neutralamino acid by another neutral amino acid.

Methods of Engineering Antibodies

Antibodies having VH and VL sequences disclosed herein may be used tocreate new variant antibodies by modifying the VH and/or VL sequences,or the constant region(s) attached thereto. Thus, the structuralfeatures of an variant antibody of the invention, are used to createstructurally related variant antibodies that retain at least onefunctional property of the antibodies of the invention, such as bindingto NPC-1 epitope peptidomimetic. The starting material for theengineering method may be one or more of the VH and/or VK sequencesprovided herein, or one or more CDR regions thereof. To create theengineered antibody, it is not necessary to actually prepare (i.e.,express as a protein) an antibody having one or more of the VH and/or VKsequences provided herein, or one or more CDR regions thereof. Rather,the information contained in the sequence(s) is used as the startingmaterial to create a “second generation” sequence(s) derived from theoriginal sequence(s) and then the “second generation” sequence(s) isprepared and expressed as a protein. Standard molecular biologytechniques may be used to prepare and express altered antibody sequence.

Mutations may be introduced randomly or selectively along all or part ofan antibody coding sequence and the resulting modified antibodies may bescreened for binding activity and/or other desired functionalproperties. See WO 2002/092780 and WO 2003/074679.

Nucleic Acids Encoding the NPC-1 Epitope Peptidomimetic

Another aspect of the invention pertains to nucleic acid molecules thatencode the NPC-1 epitope peptidomimetic. The nucleic acids may bepresent in whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid may be isolated by purificationaway from other cellular components or other contaminants (e.g., othercellular nucleic acids or proteins) by standard techniques, includingalkaline/SDS treatment, CsC1 banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See Ausubel, et al.(2011) Current Protocols in Molecular Biology John Wiley & Sons, Inc. Anucleic acid of the invention may be, for example, DNA or RNA and may ormay not contain intronic sequences. The nucleic acid may be a cDNAmolecule.

Nucleic acids of the invention may be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma may be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody may be recovered fromthe library.

Specifically, degenerate codon substitutions may be achieved bygenerating, e.g., sequences in which the third position of one or moreselected codons is substituted with mixed-base and/or deoxyinosineresidues. Batzer, et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka, etal. (1985) J. Biol. Chem. 260: 2605-08; Rossolini, et al. (1994) Mol.Cell. Probes 8: 91-98.

Methods of Producing Recombinantly Producing NPC-1 EpitopePeptidomimetics

The present invention also provides methods for recombinantly producingthe NPC-1 epitope peptidomimetic. Methods of producing peptidomimeticsare well known to those of ordinary skill in the art.

NPC-1 epitope peptidomimetics of the invention may also be produced byconstructing, using conventional techniques well known to those ofordinary skill in the art, an expression vector containing an operon anda DNA sequence encoding the NPC-1 epitope peptidomimetics. Furthermore,the invention relates to vectors, especially plasmids, cosmids, viruses,bacteriophages and other vectors common in genetic engineering, whichcontain the above-mentioned nucleic acid molecules of the invention. Thenucleic acid molecules contained in the vectors may be linked toregulatory elements that ensure the transcription in prokaryotic andeukaryotic cells.

Vectors contain elements that facilitate manipulation for the expressionof a foreign protein within the target host cell. Conveniently,manipulation of sequences and production of DNA for transformation isfirst performed in a bacterial host (e.g., E. coli) and usually vectorswill include sequences to facilitate such manipulations, including abacterial origin of replication and appropriate bacterial selectionmarker. Selection markers encode proteins necessary for the survival orgrowth of transformed host cells grown in a selective culture medium.Host cells not transformed with the vector containing the selection genewill not survive in the culture medium. Typical selection genes encodeproteins that confer resistance to antibiotics or other toxins,complement auxotrophic deficiencies, or supply critical nutrients notavailable from complex media. Exemplary vectors and methods fortransformation of yeast are described in the art. See, e.g., Burke, etal. (2000) Methods in Yeast Genetics Cold Spring Harbor LaboratoryPress.

The polypeptide coding sequence of NPC-1 epitope peptidomimetics may beoperably linked to transcriptional and translational regulatorysequences that provide for expression of the polypeptide in yeast cells.These vector components may include, but are not limited to, one or moreof the following: an enhancer element, a promoter, and a transcriptiontermination sequence. Sequences for the secretion of the polypeptide mayalso be included (e.g., a signal sequence).

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” refers broadly to contiguous linked DNA sequences,and, in the case of a secretory leader, contiguous and in reading frame.However, enhancers do not have to be contiguous.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequences to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters(e.g., that increase levels of transcription in response to absence of arepressor). Inducible promoters may initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions (e.g., the presence or absence of a nutrient or achange in temperature.)

The expression vectors are transfected into a host cell by conventiontechniques well known to those of ordinary skill in the art to produce atransfected host cell, said transfected host cell cultured byconventional techniques well known to those of ordinary skill in the artto produce said NPC-1 epitope peptidomimetics.

The host cells used to express the NPC-1 epitope peptidomimetics may beeither a bacterial cell such as E. coli, yeast (e.g., S. cerevisiae), ora eukaryotic cell (e.g., a mammalian cell line). A mammalian cell of awell-defined type for this purpose, such as a myeloma cell, 3T3, HeLa,C6A2780, Vero, MDCK II, a Chinese hamster ovary (CHO), Sf9, Sf21, COS,NSO, or HEK293 cell line may be used.

The general methods by which the vectors may be constructed,transfection methods required to produce the host cell and culturingmethods required to produce the antibodies, and fragments thereof, fromsaid host cells all include conventional techniques. Although preferablythe cell line used to produce the NPC-1 epitope peptidomimetics is amammalian cell line, any other suitable cell line, such as a bacterialcell line such as an E. coli-derived bacterial strain, or a yeast cellline, may be used.

Similarly, once produced the NPC-1 epitope peptidomimetics may bepurified according to standard procedures in the art, such as forexample cross-flow filtration, ammonium sulphate precipitation, andaffinity column chromatography.

Generation of Monoclonal Antibodies that Bind a NPC-1 EpitopePeptidomimetic Using Animals

Antibodies that selectively bind NPC-1 epitope peptidomimetics may behuman monoclonal antibodies. Such human monoclonal antibodies directedagainst a NPC-1 epitope peptidomimetic may be generated using transgenicor transchromosomic mice carrying parts of the human immune systemrather than the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse® and KM Mouse®respectively, and are collectively referred to herein as “human Igmice.” The HuMAb Mouse® (Medarex. Inc.) contains human immunoglobulingene miniloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci. See, e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859. Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal. Lonberg (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg and Huszar (1995) Intern. Rev. Immunol. 13: 65-93, andHarding and Lonberg (1995) Ann. NY. Acad. Sci. 764: 536-546. Thepreparation and use of the HuMab Mouse®, and the genomic modificationscarried by such mice, is further described in Taylor, et al. (1992)Nucleic Acids Research 20: 6287-6295; Chen, et al. (1993) InternationalImmunology 5: 647-656; Tuaillon, et al. (1993) Proc. Natl. Acad. Sci.USA 90: 3720-3724; Choi, et al. (1993) Nature Genetics 4: 117-123; Chen,et al. (1993) EMBO J. 12: 821-830; Tuaillon, et al. (1994) J. Immunol.152: 2912-2920; Taylor, et al. (1994) International Immunology 6:579-591; and Fishwild, et al. (1996) Nature Biotechnology 14: 845-851.See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and5,545,807; WO 92/03918, WO 93/12227, WO 94/25585; WO 97/13852; WO98/24884; WO 99/45962; and WO 01/14424.

Human antibodies that selectively bind the NPC-1 epitope peptidomimeticsof the invention may be raised using a mouse that carries humanimmunoglobulin sequences on transgenes and transchromosomes, such as amouse that carries a human heavy chain transgene and a human light chaintranschromosome. Such mice, referred to herein as “KM Mice®”, aredescribed in detail in WO 02/43478.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and may be used to raiseantibodies that selectively bind NPC-1 epitope peptidomimetics. Forexample, an alternative transgenic system referred to as the Xenomouse(Abgenix, Inc.) may be used; such mice are described in, for example,U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and may be used to raiseantibodies that selectively bind NPC-11 epitope peptidomimetics. Forexample, mice carrying both a human heavy chain transchromosome and ahuman light chain transchromosome, referred to as “TC mice” may be used.See Tomizuka, et al. (2000) Proc. Natl. Acad Sci. USA 97: 722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (Kuroiwa, et al. (2002) NatureBiotechnology 20: 889-894) and may be used to raise antibodies thatselectively bind NPC-1 epitope peptidomimetics.

Human monoclonal antibodies of the invention may also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See, for example, U.S. Pat. Nos. 5,223,409;5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197;5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

Human monoclonal antibodies of the invention may also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response may be generated upon immunization. See,e.g., U.S. Pat. Nos. 5,476,996 and 5,698,767.

When human Ig mice are used to raise human antibodies of the invention,such mice may be immunized with a purified or enriched preparation ofNPC-1 epitope peptidomimetic, as described by Lonberg, et al. (1994)Nature 368(6474): 856-859; Fishwild, et al. (1996) Nature Biotechnology14: 845-851; WO 98/24884 and WO 01/14424. Preferably, the mice will be6-16 weeks of age upon the first infusion. For example, a purified orrecombinant preparation (5-50 μg) of NPC-1 epitope may be used toimmunize the human Ig mice intraperitoneally.

Prior experience with various antigens by others has shown that thetransgenic mice respond when initially immunized intraperitoneally (IP)with antigen in complete Freund's adjuvant, followed by every other weekIP immunizations (up to a total of 6) with antigen in incompleteFreund's adjuvant. However, adjuvants other than Freund's are also foundto be effective. In addition, whole cells in the absence of adjuvant arefound to be highly immunogenic. The immune response may be monitoredover the course of the immunization protocol with plasma samples beingobtained by retroorbital bleeds. The plasma may be screened by ELISA (asdescribed below), and mice with sufficient titers of anti-NPC-1 humanimmunoglobulin may be used for fusions. Mice may be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen. It is expected that 2-3 fusions for each immunization may needto be performed. Between 6 and 24 mice are typically immunized for eachantigen. Usually both HCo7 and HCo12 strains are used. In addition, bothHCo7 and HCo12 transgene may be bred together into a single mouse havingtwo different human heavy chain transgenes (HCo7/HCo12). Alternativelyor additionally, the KM Mouse® strain may be used.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice maybe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas may be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice may be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells may be plated at approximately2×10⁻⁵ in flat bottom microtiter plate, followed by a two weekincubation in selective medium containing 20% fetal Clone Serum, 18%“653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodiumpyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/mlpenicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma;the HAT is added 24 hours after the fusion). After approximately twoweeks, cells may be cultured in medium in which the HAT is replaced withHT. Individual wells may then be screened by ELISA for human monoclonalIgM and IgG antibodies. Once extensive hybridoma growth occurs, mediummay be observed usually after 10-14 days. The antibody secretinghybridomas may be replated, screened again, and if still positive forhuman IgG, the monoclonal antibodies may be subcloned at least twice bylimiting dilution. The stable subclones may then be cultured in vitro togenerate small amounts of antibody in tissue culture medium forcharacterization.

To purify human monoclonal antibodies, selected hybridomas may be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants may be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.)Eluted IgG may be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution may beexchanged into PBS, and the concentration may be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies may bealiquoted and stored at −80° C.

Polynucleotides Encoding NPC-1 Epitope Peptidomimetics

The present invention also provides MUC5AC antigen nucleotides whichencode NPC-1 epitope peptidomimetics. The present invention alsoprovides for fragments, sequences hybridizable with, and sequenceshomologous to the polynucleotide sequences that encode a NPC-1 epitopepeptidomimetic which are at least about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%,99.9%, or 100%.

The invention also provides polynucleotides comprising at least oneNPC-1 epitope peptidomimetic sequence encoding similar polypeptides withdifferent codon usage, altered sequences characterized by mutations,such as deletion, insertion or substitution of one or more nucleotides,either naturally occurring or man induced, either randomly or in atargeted fashion. The present invention also encompasses homologousnucleic acid sequences (e.g., which form a part of a polynucleotidesequence of the present invention), which include sequence regionsunique to the polynucleotides of the present invention.

The present invention also encompasses nucleic acids encoding homologuesof NPC-1 epitope peptidomimetics, such homologues can be at least about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identicalhomologous to the amino acid sequences set forth herein, as may bedetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. The present invention alsoencompasses fragments of the above described polynucleotides andpolypeptides having mutations, such as deletions, insertions orsubstitutions of one or more nucleic acids, either naturally occurringor man induced, either randomly or in a targeted fashion.

Nucleic acid molecules may encode a NPC-1 epitope peptidomimetic of saidnucleic acid molecule. A “functional fragment” of said nucleic acidincludes a fragment of the gene or cDNA encoding said NPC-1 epitopepeptidomimetic, which fragment is capable of being expressed to producea NPC-1 epitope capable of eliciting an immune response (e.g.,antibodies which selectively bind the NPC-1 epitope) Thus, for example,fragments of the NPC-1 epitope peptidomimetic according to the inventionwhich correspond to amino acid residues that contribute to theimmunogenicity of the antigen and which fragments may serve to functionas antigens to elicit an immune response (e.g., humoral or cellularimmune response.) This aspect of the invention also includesdifferentially spliced isoforms and transcriptional starts of thenucleic acids according to the invention. The nucleic acid moleculesaccording to the invention also comprise fragments, derivatives andallelic variants of the nucleic acid molecules described above thatencodes a NPC-1 epitope peptidomimetic according to the invention.Methods and materials for making nucleic acids encoding fragments ofNPC-1 epitope peptidomimetics are well known in the art. See, e.g.,Maniatis, et al. (2001) Molecular Cloning: A Laboratory Manual [3^(rd)Ed.] Cold Spring Harbor Laboratory Press.

Furthermore, identity refers broadly to the that functional and/orstructural equivalence that exists between the nucleic acid moleculesconcerned or the proteins coded by them. The nucleic acid molecules,which are homologous to the molecules described above and constitutederivatives of these molecules, are generally variations of thesemolecules, which constitute modifications, which execute the samebiological function. At the same time, the variations may occurnaturally, for example they may be sequences from other species, or theymay be mutants, wherein these mutants may have occurred in a naturalmanner or have been introduced by objective mutagenesis. The variationsmay also be synthetically manufactured sequences. The allelic variantsmay be both naturally occurring variants and also syntheticallymanufactured variants or variants produced by recombinant DNAtechniques. Nucleic acid molecules, which deviate from nucleic acidmolecules according to the invention due to degeneration of the geneticcode, constitute a special form of derivatives.

Included also within the scope of the invention is any nucleotidesequence that encodes the amino acid sequence of NPC-1 epitopepeptidomimetics thereof. Because the genetic code is degenerate, morethan one codon may be used to encode a particular amino acid. Using thegenetic code, one or more different nucleotides may be identified, eachof which would be capable of encoding the amino acid. The probabilitythat a particular nucleotide will, in fact, constitute the actual codonencoding sequence may be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic orprokaryotic cells expressing a NPC-1 epitope peptidomimetic thereof.Such “codon usage rules” are disclosed by Lathe, et al. (1985) J. Molec.Biol. 183: 1-12.

Modified NPC-1 Epitope Peptidomimetics

The nucleotides of the present invention may be modifiedpolynucleotides. Unmodified nucleotide are often less optimal in someapplications, e.g., prone to degradation by cellular nucleases. Chemicalmodifications to one or more of the subunits of oligonucleotide mayconfer improved properties, e.g., may render polynucleotides more stableto nucleases. Typical oligonucleotide modifications are well-known inthe art and may include one or more of: (i) alteration, e.g.,replacement, of one or both of the non-linking phosphate oxygens and/orof one or more of the linking phosphate oxygens in the phosphodiesterintersugar linkage; (ii) alteration, e.g., replacement, of a constituentof the ribose sugar, e.g., of the modification or replacement of the 2′hydroxyl on the ribose sugar, (iii) wholesale replacement of thephosphate moiety; (iv) modification or replacement of a naturallyoccurring base with a non-natural base; (v) replacement or modificationof the ribose-phosphate backbone, e.g. with peptide nucleic acid (PNA);(vi) modification of the 3′ end or 5′ end of the oligonucleotide; and(vii) modification of the sugar, e.g., six membered rings.Polynucleotides used in accordance with this invention may besynthesized by any number of means well-known in the art, or purchasedfrom a variety of commercial vendors (LC Sciences, Houston, Tex.;Promega, Madison, Wis.; Invitrogen, Carlsbad, Calif.).

Isolation and expression of the NPC-1 epitope peptidomimetic, orfragments and variants thereof, of the invention may be effected bywell-established cloning procedures using probes or primers constructedbased on the NPC-1 epitope peptidomimetic nucleic acids sequencesdisclosed in the application. Related NPC-1 epitope peptidomimeticsequences may also be identified from human or other species genomicdatabases using the sequences disclosed herein and known computer-basedsearch technologies, e.g., BLAST sequence searching. The pseudogenesdisclosed herein may be used to identify functional alleles or relatedgenes.

Expression vectors can then be used to infect or transfect host cellsfor the functional expression of these sequences. These genes andvectors can be made and expressed in vitro or in vivo. One of skill willrecognize that desired phenotypes for altering and controlling nucleicacid expression can be obtained by modulating the expression or activityof the genes and nucleic acids (e.g., promoters, enhancers) within thevectors of the invention. Any of the known methods described forincreasing or decreasing expression or activity can be used.

The polynucleotide sequences provided herein may be generated accordingto any oligonucleotide synthesis method known in the art such asenzymatic synthesis or solid phase synthesis. Equipment and reagents forexecuting solid-phase synthesis are commercially available from, forexample, Applied Biosystems. Any other means for such synthesis may alsobe employed; the actual synthesis of the polynucleotides is well withinthe capabilities of one skilled in the art. See, e.g., Maniatis, et al.(2001) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.] Cold SpringHarbor Laboratory Press; Swamy (2008) Laboratory Manual on BiotechnologyRastogi Publications; Herdewijn (2005) [Ed.] Methods in MolecularBiolog: Oligonucleotide Synthesis: Methods and Applications Volume 288Humana Press; and Rapley (2000) [Ed.] The Nucleic Acid ProtocolsHandbook Humana Press. Double-stranded DNA fragments may then beobtained either by synthesizing the complementary strand and annealingthe strands together under appropriate conditions, or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

Techniques for the manipulation of nucleic acids, such as, for example,for generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization are well described in the scientific andpatent literature. See, e.g., Sambrook, et al. (2001) (Eds.) MolecularCloning A Laboratory Manual (3^(rd) Ed.) Cold Spring Harbor Laboratory;Ausubel, et al. (2011) Ed., Current Protocols in Molecular Biology. JohnWiley & Sons, Inc., New York; Tijssen (1993) [Ed.] Laboratory Techniquesin Biochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I, Theory and Nucleic Acid Preparation, Elsevier, N.Y.

Hybridization and the strength of hybridization (e.g., the strength ofthe association between polynucleotides) is impacted by many factorswell known in the art including the degree of complementarity betweenthe polynucleotides, and the stringency of the conditions involved,which is affected by such conditions as the concentration of salts, thepresence of other components (e.g., the presence or absence ofpolyethylene glycol), the molarity of the hybridizing strands and theG+C content of the polynucleotide strands, all of which results in acharacteristic melting temperature (T_(m)) of the formed hybrid.Techniques of nucleic acid hybridization are disclosed by Sambrook, etal. (2001) (Eds.) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.]Cold Spring Harbor Laboratory, and by Hayrnes, et al. (1985) in NUCLEICACID HYBRIDIZATION. A PRACTICAL APPROACH (IRL Press, DC). Hybridizationwash conditions may include wash solution of 0.2×SSC/0.1% SDS andincubation with rotation for 10 minutes at room temperature, (lowstringency wash), wash solution of prewarmed (42° C.) 0.2×SSC/0.1% SDSand incubation with rotation for 15 minutes at 42° C. (medium stringencywash) and wash solution of prewarmed (68° C.) 0.1×SSC/0.1% SDS andincubation with rotation for 15 minutes at 68° C. (high stringencywash). See Ausubel, et al. (2011) [Ed.] Current Protocols in MolecularBiology John Wiley & Sons, Inc.

Oligonucleotide primers may be used to amplify nucleic acids encoding aNPC-1 epitope peptidomimetics. The nucleic acids described herein canalso be cloned or measured quantitatively using amplificationtechniques. Amplification methods are also well known in the art, andinclude, e.g., polymerase chain reaction (PCR) (Innis (1990) [Ed.] PCRProtocols, a Guide to Methods and Applications, Academic Press, NY.;Innis (1995) [Ed.] PCR Strategies. Academic Press, Inc., NY.); ligasechain reaction (LCR) (Wu (1989) Genomics 4: 560; Landegren (1988)Science 241: 1077; Barringer (1990) Gene 89: 117); transcriptionamplification (Kwoh (1989) PNAS 86: 1173); self-sustained sequencereplication (Guatelli (1990) PNAS 87: 1874); Q Beta replicaseamplification (Smith (1997) J. Clin. Microbiol. 35: 1477-91)); automatedQ-beta replicase amplification assay (Burg (1996) Mol. Cell. Probes 10:257-71); and other RNA polymerase mediated techniques (e.g., NASBA,Cangene, Mississauga, Ontario). See, also, Berger (1987) MethodsEnzymol. 152: 307-16; Sambrook, et al. (2001) (Eds.) Molecular Cloning:A Laboratory Manual (3^(rd) Ed.) Cold Spring Harbor Laboratory; Ausubel,et al. (2011) [Ed.] Current Protocols in Molecular Biology. John Wiley &Sons, Inc., New York; Maniatis, et al. (2001) Molecular Cloning: ALaboratory Manual [3^(rd) Ed.] Cold Spring Harbor Laboratory Press; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-64.

Paradigms to design degenerate primer pairs are well known in the art.For example, a Consensus Degenerate Hybrid Oligonucleotide Primer(CODEHOP) strategy computer program is readily accessible and isdirectly linked from the BlockMaker multiple sequence alignment site forhybrid primer prediction beginning with a set of related proteinsequences, such as the NPC-1 epitope peptidomimetic sequences providedherein. See, e.g., Rose (1998) Nucleic Acids Res. 26: 1628-35; Singh(1998) Biotechniques 24: 318-19.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to NPC-1 epitopes disclosed herein may beisolated using the nucleic acid probes described above. Alternatively,expression libraries can be used to clone NPC-1 epitope peptidomimeticsand polymorphic variants, alleles, and interspecies homologs thereof, bydetecting expressed homologs immunologically with antisera or purifiedantibodies made against a NPC-1 epitope peptidomimetic, which alsorecognize and selectively bind to the NPC-1 epitope peptidomimetichomolog.

Nucleic acids that encode NPC-1 epitope peptidomimetics may be generatedby amplification (e.g., PCR) of appropriate nucleic acid sequences usingappropriate (perfect or degenerate) primer pairs. The amplified nucleicacid can be genomic DNA from any cell or tissue or mRNA or cDNA derivedfrom NPC-1 expressing cells. Methods for expression of heterologoussequences in host cells are well known in the art. See, e.g., Maniatis,et al. (2001) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.] ColdSpring Harbor Laboratory Press.

Fusion Proteins Comprising a NPC-1 Epitope Peptidomimetics

Hybrid protein-coding sequences comprising nucleic acids encoding NPC-1epitope peptidomimetics fused to a translocation sequences may beconstructed. Also provided are hybrid NPC-1 epitopes comprising themotifs and antigenic regions. These nucleic acid sequences may beoperably linked to transcriptional or translational control elements,e.g., transcription and translation initiation sequences, promoters andenhancers, transcription and translation terminators, polyadenylationsequences, and other sequences useful for transcribing DNA into RNA. Inconstruction of recombinant expression cassettes, vectors, andtransgenics, a promoter fragment can be employed to direct expression ofthe desired nucleic acid in all desired cells or tissues.

Fusion proteins may comprise C-terminal or N-terminal translocationsequences. Further, fusion proteins can comprise additional elements,e.g., for protein detection, purification, or other applications.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts, histidine-tryptophanmodules, or other domains that allow purification on immobilized metals;maltose binding protein; protein A domains that allow purification onimmobilized immunoglobulin; or the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.)

The inclusion of a cleavable linker sequences such as Factor Xa (see,e.g., Ottavi, (1998) Biochimie 80: 289-93), subtilisin proteaserecognition motif (see, e.g., Polyak (1997) Protein Eng. 10: 615-19);enterokinase (Invitrogen, San Diego, Calif.), between the translocationdomain (for efficient plasma membrane expression) and the rest of thenewly translated polypeptide may be useful to facilitate purification.For example, one construct can include a polypeptide encoding a nucleicacid sequence linked to six histidine residues followed by athioredoxin, an enterokinase cleavage site (see, e.g., Williams (1995)Biochemistry 34: 1787-97), and an C-terminal translocation domain. Thehistidine residues facilitate detection and purification while theenterokinase cleavage site provides a means for purifying the desiredprotein(s) from the remainder of the fusion protein. Technologypertaining to vectors encoding fusion proteins and application of fusionproteins are well described in the scientific and patent literature.See, e.g., Kroll (1993) DNA Cell. Biol. 12: 441-53.

Systems for Recombinant Expression of the NPC-1 Epitope Peptidomimetics

Expression vectors, either as individual expression vectors or aslibraries of expression vectors, comprising the ligand-binding regionencoding sequences may be introduced into a genome or into the cytoplasmor a nucleus of a cell and expressed by a variety of conventionaltechniques, well described in the scientific and patent literature. See,e.g., Sambrook, et al. (2001) [Eds.] Molecular Cloning: A LaboratoryManual (3^(rd) Ed.) Cold Spring Harbor Laboratory; Ausubel, et al.(2011) [Ed.] Current Protocols in Molecular Biology John Wiley & Sons,Inc.

The nucleic acids can be expressed in expression cassettes, vectors orviruses which are stably or transiently expressed in cells (e.g.,episomal expression systems). Selection markers can be incorporated intoexpression cassettes and vectors to confer a selectable phenotype ontransformed cells and sequences. For example, selection markers can codefor episomal maintenance and replication such that integration into thehost genome is not required. For example, the marker may encodeantibiotic resistance (e.g., chloramphenicol, kanamycin, 0418,bleomycin, hygromycin) or herbicide resistance (e.g., chlorosulfurone orBasta) to permit selection of those cells transformed with the desiredDNA sequences. See, e.g., Ausubel, et al. (2011) [Ed.] Current Protocolsin Molecular Biology John Wiley & Sons, Inc.; and Walker & Papley (2009)Molecular Biology and Biotechnology [5^(th) Ed.] Royal Society ofChemistry. Because selectable marker genes conferring resistance tosubstrates like neomycin or hygromycin can only be utilized in tissueculture, chemoresistance genes are also used as selectable markers invitro and in vivo.

To enable cellular expression of the polynucleotides of the presentinvention, a nucleic acid construct according to the present inventionmay be used, which includes at least a coding region of one of the abovenucleic acid sequences, and further includes at least one cis actingregulatory element Preferably, the promoter utilized by the nucleic acidconstruct of the present invention is active in the specific cellpopulation transformed. Examples of cell type-specific and/ortissue-specific promoters are well-known in the art. See Bernardi (2003)[Ed.] Gene Transfer and Expression in Mammalian Cells Volume 38 ElsevierScience B.V. The nucleic acid construct of the present invention canfurther include an enhancer, which can be adjacent or distant to thepromoter sequence and can function in up regulating the transcriptiontherefrom.

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible for propagation in cells, or integrationin a gene and a tissue of choice. The construct according to the presentinvention can be, for example, a plasmid, a bacmid, a phagemid, acosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3,pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto,pCMV/myc/cyto each of which is commercially available from InvitrogenCo. (Carlsbad, Calif.) Examples of retroviral vector and packagingsystems are those sold by Clontech (San Diego, Calif.), includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiplecloning sites and the transgcnc is transcribed from CMV promoter.Vectors derived from Mo-MuLV are also included such as pBabe, where thetransgene will be transcribed from the 5′ LTR promoter.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Regulatory sequences include thosethat direct constitutive expression of a nucleotide sequence in manytypes of host cell and those that direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention may be designed forproduction of variant proteins in prokaryotic or eukaryotic cells. Forexample, proteins of the invention can be expressed in bacterial cellssuch as Escherichia coli, insect cells (e.g., using baculovirusexpression vectors), yeast cells, or mammalian cells. Suitable hostcells are discussed further in Goeddel (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, to the amino or C terminus of the recombinant protein.Such fusion vectors typically serve three purposes: (i) to increaseexpression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin,PreScission, TEV and enterokinase. Typical fusion expression vectorsinclude pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

The recombinant mammalian expression vector is capable of directingexpression of the nucleic acid may be in a particular cell type (e.g.,tissue-specific regulatory elements are used to express the nucleicacid). Tissue-specific regulatory elements are known in the art. Forefficient production of the protein, it is preferable to place thenucleotide sequences encoding the protein of the invention under thecontrol of expression control sequences optimized for expression in adesired host. For example, the sequences may include optimizedtranscriptional and/or translational regulatory sequences (e.g., alteredKozak sequences).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman(1990) Gene Expression Technology: Methods in Enzymology Academic Press,San Diego, Calif. 185: 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli. See, e.g., Wada, et al. (1992) Nucl.Acids Res. 20: 2111-2118. Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.Another strategy to solve codon bias is by using BL21-codon plusbacterial strains (Invitrogen) or Rosetta bacterial strain (Novagen),these strains contain extra copies of rare E. coli tRNA genes.

The expression vector encoding for the protein of the invention may be ayeast expression vector. Examples of vectors for expression in yeastSaccharomyces cerevisiae include pYepSec1 (Baldari, et al. (1987) EMBOJ. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943),pJRY88 (Schultz, et al. (1987) Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego,Calif.)

Alternatively, polypeptides of the present invention can be produced ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39). In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed (1987)Nature 329: 840) and pMT2PC (Kaufman, et al. (1987) EMBO J. 6: 187-195),pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4(Invitrogen), pcDNA3 (Invitrogen). When used in mammalian cells, theexpression vector's control functions are often provided by viralregulatory elements. For example, commonly used promoters are derivedfrom polyoma, adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Sambrook, et al. (2001)(Eds.) Molecular Cloning: A Laboratory Manual (3^(rd) Ed.) Cold SpringHarbor Laboratory.

A host cell can be any prokaryotic or eukaryotic cell. For example,protein of the invention can be produced in bacterial cells such as E.coli, insect cells, yeast, plant or mammalian cells (e.g., ChineseHamster Ovary cells (CHO), COS, HEK293 cells). Other suitable host cellsare known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (2001)[Eds.]Molecular Cloning: A Laboratory Manual (3^(rd) Ed.) Cold SpringHarbor Laboratory and other laboratory manuals.

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell. See,e.g., Sambrook, et al. (2001) (Eds.) Molecular Cloning: A LaboratoryManual (3^(rd) Ed.) Cold Spring Harbor Laboratory and Walker & Papley(2009) Molecular Biology and Biotechnology [5^(rd) Ed.] Royal Society ofChemistry. It is only necessary that the particular genetic engineeringprocedure used be capable of successfully introducing at lest onenucleic acid molecule into the host cell capable of expressing the NPC-1epitope peptidomimetic.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin, puromycin, blasticidin, and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding protein of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) protein of theinvention. Accordingly, the invention further provides methods forproducing proteins of the invention using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the present invention (into which a recombinant expressionvector encoding protein of the invention has been introduced) in asuitable medium such that the protein of the invention is produced. Inanother embodiment, the method further comprises isolating protein ofthe invention from the medium or the host cell.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe receptor, fragment, or variant of interest, which is then recoveredfrom the culture using standard techniques. Examples of such techniquesare well known in the art. See, e.g., WO 00/06593.

Labels

The NPC-1 epitope peptidomimetics described herein may be modifiedpost-translationally to add effector moieties such as chemical linkers,detectable moieties such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials,chemiluminescent moieties, a cytotoxic agent, radioactive materials, orfunctional moieties.

A wide variety of entities, e.g., ligands, may be coupled to theoligonucleotides as known in the art. Ligands may include naturallyoccurring molecules, or recombinant or synthetic molecules. Exemplaryligands include, but are not limited to, avadin, biotin, peptides,peptidomimetics, polylysine (PLL), polyethylene glycol (PEG), mPEG,cationic groups, spermine, spermidine, polyamine, thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, mucin,glycosylated polyaminoacids, transferrin, aptamer, immunoglobulins(e.g., antibodies), insulin, transferrin, albumin, sugar, lipophilicmolecules (e.g., steroids, bile acids, cholesterol, cholic acid, andfatty acids), vitamin A, vitamin E, vitamin K, vitamin B, folic acid,B12, riboflavin, biotin, pyridoxal, vitamin cofactors,lipopolysaccharide, hormones and hormone receptors, lectins,carbohydrates, multivalent carbohydrates, radiolabeled markers,fluorescent dyes, and derivatives thereof. See, e.g., U.S. Pat. Nos.6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437;6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559, 279.

Additionally, moieties may be added to the NPC-1 epitope peptidomimeticto increase half-life in vivo (e.g., by lengthening the time toclearance from the blood stream. Such techniques include, for example,adding PEG moieties (also termed pegilation), and are well-known in theart. See U.S. Patent Application Publication No. 2003/0031671.

An peptidomimetic, antibody or antigen binding fragment thereof,described herein may be “attached” to a substrate when it is associatedwith the solid label through a non-random chemical or physicalinteraction. The attachment may be through a covalent bond. However,attachments need not be covalent or permanent. Materials may be attachedto a label through a “spacer molecule” or “linker group.” Such spacermolecules are molecules that have a first portion that attaches to thebiological material and a second portion that attaches to the label.Thus, when attached to the label, the spacer molecule separates thelabel and the biological materials, but is attached to both. Methods ofattaching biological material (e.g., label) to a label are well known inthe art, and include but are not limited to chemical coupling.

Detectable Labels

The NPC-1 epitope peptidomimetic described herein may be modifiedpost-translationally to add effector labels such as chemical linkers,detectable labels such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent labels, or functional labels such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials. Further exemplary enzymes include, but are notlimited to, horseradish peroxidase, acetylcholinesterase, alkalinephosphatase, β-galactosidase and luciferase. Further exemplaryfluorescent materials include, but are not limited to, rhodamine,fluorescein, fluoresccin isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin and dansyl chloride. Furtherexemplary chemiluminescent labels include, but are not limited to,luminol. Further exemplary bioluminescent materials include, but are notlimited to, luciferin and aequorin. Further exemplary radioactivematerials include, but are not limited to, bismuth-213 (²¹³Bs),carbon-14 (¹⁴C), carbon-11 (¹¹C), chlorine-18 (Cl¹⁸), chromium-51(⁵¹Cr), cobalt-57 (⁵⁷Co), cobalt-60 (⁶⁰Co), copper-64 (⁶⁴Cu), copper-67(⁶⁷Cu), dysprosium-165 (¹⁶⁵Dy), erbium-169 (¹⁶⁹Er), fluorine-18 (¹⁸F),gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga), germanium-68 (⁶⁸Ge), holmium-166(¹⁶⁶Ho), indium-111 (¹¹¹In), iodine-125 (¹²⁵I), iodine-123 (¹²⁴I),iodine-124 (¹²⁴I), iodine-131 (¹³¹I), iridium-192 (¹⁹²Ir), iron-59(⁵⁰Fe), krypton-81 (⁸¹Kr), lead-212 (²¹²Pb), lutetium-177 (¹⁷⁷Lu),molybdenum-99 (⁹⁹Mo), nitrogen-13 (¹³N), oxygen-15 (¹⁵O), palladium-103(¹⁰³Pd), phosphorus-32 (³²P), potassium-42 (⁴²K), rhenium-186 (¹⁸⁶Re),rhenium-188 (¹⁸⁸Re), rubidium-81 (⁸¹Rb), rubidium-82 (⁸²Rb),samarium-153 (¹⁵³Sm), selenium-75 (⁷⁵Se), sodium-24 (²Na), strontium-82(⁸²Sr), strontium-89 (⁸⁹Sr), sulfur 35 (³⁵S), technetium-99m (⁹⁹Tc),thallium-201 (²⁰¹Tl), tritium (3H), xenon-133 (¹³³Xe), ytterbium-169(¹⁶⁹Yb), ytterbium-177 (¹⁷⁷Yb), and yttrium-90 (⁹⁰Y).

Cytotoxic Agents

The NPC-1 epitope peptidomimetic described herein may be conjugated tocytotoxic agents including, but are not limited to, methotrexate,aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine; alkylating agents such as mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine(CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine,busulfan, dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin(paraplatin); anthracyclines include daunorubicin (formerly daunomycin),doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin,epirubicin, mitoxantrone and bisantrene; antibiotics includedactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, andanthramycin (AMC); and antimytotic agents such as the vinca alkaloids,vincristine and vinblastine. Other cytotoxic agents include paclitaxel(TAXOL), ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B,gramicidin D, ethidium bromide, emetine, etoposide, tenoposide,colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxicagents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGFantagonists, EGFR antagonists, platins, taxols, irinotecan,5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide,melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesineand vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy,sex hormone antagonists, selective androgen receptor modulators,selective estrogen receptor modulators, PDGF antagonists, TNFantagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12Rantagonists, Toxin conjugated monoclonal antibodies, tumor antigenspecific monoclonal antibodies, Erbitux®, Avastin®, Pertuzumab,anti-CD20 antibodies, Rituxan®, ocrelizumab, ofatumumab, DXL625,Herceptin®, or any combination thereof. Toxic enzymes from plants andbacteria such as ricin, diphtheria toxin and Pseudomonas toxin may beconjugated to the humanized antibodies, or binding fragments thereof, togenerate cell-type-specific-killing reagents. Youle, et al. (1980) Proc.Nat'l Acad. Sci. USA 77: 5483; Gilliland, et al. (1980) Proc. Nat'lAcad. Sci. USA 77: 4539; Krolick, et al. (1980) Proc. Nat'l Acad. Sci.USA 77: 5419. Other cytotoxic agents include cytotoxic ribonucleases.See U.S. Pat. No. 6,653,104.

The NPC-1 epitope peptidomimetics described herein may be conjugated toa radionuclide that emits alpha or beta particles (e.g.,radioimmunoconjuagtes). Such radioactive isotopes include but are notlimited to beta-emitters such as phosphorus-32 (³²P), scandium-47(⁴⁷Sc), copper-67 (⁶⁷Cu), gallium-67 (⁶⁷Ga), yttrium-88 (⁸⁸Y),yttrium-90 (⁹⁰Y), iodine-125 (¹²⁵I), iodine-131 (¹³¹I), samarium-153(¹⁵³Sm), lutetium-177 (¹⁷⁷Lu), rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re),and alpha-emitters such as astatine-211 (²¹¹At), lead-212 (²¹²Pb),bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi) or actinium-225 (²²⁵Ac).

Methods are known in the art for conjugating a NPC-1 epitopepeptidomimetics described herein to a label, such as those methodsdescribed by Hunter, et al (1962) Nature 144: 945; David, et al. (1974)Biochemistry 13: 1014; Pain, et al. (1981) J. Immunol. Meth. 40: 219;and Nygren (1982) Histochem. and Cytochem. 30: 407.

Substrates

The NPC-1 epitope peptidomimetics described herein may be attached to asubstrate. A number of substrates (e.g., solid supports) known in theart are suitable for use with the NPC-1 epitope peptidomimeticsdescribed herein. The substrate may be modified to contain channels orother configurations. See Fung (2004) [Ed.] Protein Arrays: Methods andProtocols Humana Press and Kambhampati (2004) [Ed.] Protein MicroarrayTechnology John Wiley & Sons.

Substrate materials include, but are not limited to acrylics, agarose,borosilicate glass, carbon (e.g., carbon nanofiber sheets or pellets),cellulose acetate, cellulose, ceramics, gels, glass (e.g., inorganic,controlled-pore, modified, soda-lime, or functionalized glass), latex,magnetic beads, membranes, metal, metalloids, nitrocellulose, NYLON,optical fiber bundles, organic polymers, paper, plastics,polyacryloylmorpholide, poly(4-methylbutene), poly(ethyleneterephthalate), poly(vinyl butyrate), polyacrylamide, polybutylene,polycarbonate, polyethylene, polyethyleneglycol terephthalate,polyformaldehyde, polymethacrylate, polymethylmethacrylate,polypropylene, polysaccharides, polystyrene, polyurethanes,polyvinylacetate, polyvinylchloride, polyvinylidene difluoride (PVDF),polyvinylpyrrolidinone, rayon, resins, rubbers, semiconductor materials,SEPHAROSE®, silica, silicon, styrene copolymers, TEFLON®, and variety ofother polymers.

Substrates need not be flat and can include any type of shape includingspherical shapes (e.g., beads) or cylindrical shapes (e.g., fibers).Materials attached to solid supports may be attached to any portion ofthe solid support (e.g., may be attached to an interior portion of aporous solid support material).

The substrate body may be in the form of a bead, box, column, cylinder,disc, dish (e.g., glass dish, PETRI dish), fiber, film, filter,microtiter plate (e.g., 96-well microtiter plate), multi-bladed stick,net, pellet, plate, ring, rod, roll, sheet, slide, stick, tray, tube, orvial. The substrate may be a singular discrete body (e.g., a singletube, a single bead), any number of a plurality of substrate bodies(e.g., a rack of 10 tubes, several beads), or combinations thereof(e.g., a tray comprises a plurality of microtiter plates, a columnfilled with beads, a microtiter plate filed with beads).

A NPC-1 epitope peptidomimetic may be “attached” to a substrate when itis associated with the solid substrate through a non-random chemical orphysical interaction. The attachment may be through a covalent bond.However, attachments need not be covalent or permanent. Materials may beattached to a substrate through a “spacer molecule” or “linker group.”Such spacer molecules are molecules that have a first portion thatattaches to the biological material and a second portion that attachesto the substrate. Thus, when attached to the substrate, the spacermolecule separates the substrate and the biological materials, but isattached to both. Methods of attaching biological material (e.g., label)to a substrate are well known in the art, and include but are notlimited to chemical coupling.

Plates, such as microtiter plates, which support and contain thesolid-phase for solid-phase synthetic reactions may be used. Microtiterplates may house beads that are used as the solid-phase. By “particle”or “microparticle” or “nanoparticle” or “bead” or “microbead” or“microsphere” herein is meant microparticulate matter having any of avariety of shapes or sizes. The shape may be generally spherical butneed not be spherical, being, for example, cylindrical or polyhedral. Aswill be appreciated by those in the art, the particles may comprise awide variety of materials depending on their use, including, but notlimited to, cross-linked starch, dextrans, cellulose, proteins, organicpolymers including styrene polymers such as polystyrene andmethylstyrene as well as other styrene co-polymers, plastics, glass,ceramics, acrylic polymers, magnetically responsive materials, colloids,thoriasol, carbon graphite, titanium dioxide, nylon, latex, and TEFLON®.See e.g., “Microsphere Detection Guide” from Bangs Laboratories,Fishers, Ind.

The NPC-1 epitope peptidomimetics described herein may be attached to onany of the forms of substrates described herein (e.g., bead, box,column, cylinder, disc, dish (e.g., glass dish, PETRI dish), fiber,film, filter, microtiter plate (e.g., 96-well microtiter plate),multi-bladed stick, net, pellet, plate, ring, rod, roll, sheet, slide,stick, tray, tube, or vial). In particular, particles or beads may be acomponent of a gelling material or may be separate components such aslatex beads made of a variety of synthetic plastics (e.g., polystyrene).The label (e.g., streptavidin) may be bound to a substrate (e.g., bead).

Pharmaceutical Compositions

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal. Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, epicutaneous,epidural, inhalation, intraarterial, intracardial,intracerebroventricular, intradermal, intramuscular, intranasal,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, sublingual, transdermal, and transmucosal. In addition,administration may occur by means of injection, powder, liquid, gel,drops, or other means of administration.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a humanized antibody described herein, orone or more fragments thereof. The excipient generally does not provideany pharmacological activity to the formulation, though it may providechemical and/or biological stability, and release characteristics.Exemplary formulations may be found, for example, in Grennaro (2005)[Ed.] Remington: The Science and Practice of Pharmacy [21^(st) Ed.]

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition may be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriermay be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The inventionfurther contemplates the inclusion of a stabilizer in the pharmaceuticalcomposition.

The NPC-1 epitope peptidomimetics described herein may be formulatedinto pharmaceutical compositions of various dosage forms. To prepare thepharmaceutical compositions of the invention, at least one NPC-1 epitopepeptidomimetic as the active ingredient may be intimately mixed withappropriate carriers and additives according to techniques well known tothose skilled in the art of pharmaceutical formulations. See Grennaro(2005) [Ed.] Remington: The Science and Practice of Pharmacy [21^(st)Ed.] For example, the NPC-1 epitope peptidomimetics described herein maybe formulated in phosphate buffered saline pH 7.2 and supplied as a 5.0mg/mL clear colorless liquid solution.

Similarly, compositions for liquid preparations include solutions,emulsions, dispersions, suspensions, syrups, and elixirs, with suitablecarriers and additives including but not limited to water, alcohols,oils, glycols, preservatives, flavoring agents, coloring agents, andsuspending agents. Typical preparations for parenteral administrationcomprise the active ingredient with a carrier such as sterile water orparenterally acceptable oil including but not limited to polyethyleneglycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, withother additives for aiding solubility or preservation may also beincluded. In the case of a solution, it may be lyophilized to a powderand then reconstituted immediately prior to use. For dispersions andsuspensions, appropriate carriers and additives include aqueous gums,celluloses, silicates, or oils.

For each of the recited embodiments, NPC-1 epitope peptidomimetics maybe administered by a variety of dosage forms. Anybiologically-acceptable dosage form known to persons of ordinary skillin the art, and combinations thereof, are contemplated. Examples of suchdosage forms include, without limitation, reconstitutable powders,elixirs, liquids, solutions, suspensions, emulsions, powders, granules,particles, microparticles, dispersible granules, cachets, inhalants,aerosol inhalants, patches, particle inhalants, implants, depotimplants, injectables (including subcutaneous, intramuscular,intravenous, and intradermal), infusions, and combinations thereof.

In many cases, it will be preferable to include isotonic agents, e.g.,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions maybe brought about by including in the composition an agent which delaysabsorption, e.g., monostearate salts and gelatin. Moreover, thecompounds described herein may be formulated in a time releaseformulation, e.g. in a composition that includes a slow release polymer.The NPC-1 epitope peptidomimetics may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers may be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLO). Many methods for the preparation of such formulations are knownto those skilled in the art.

Optionally, adjuvants may also be included in a composition. Adjuvantswhich may be used include, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents; (3) saponin adjuvants, such asQS21 may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, interferons (e.g., gammainterferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF); (6) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT); (7) MPL or 3-O-deacylatedMPL (3dMPL); (8) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions; (9) oligonucleotides comprising CpG motifs; (10)a polyoxyethylene ether or a polyoxyethylene ester, (11) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol; (12) a saponin and an immunostimulatory oligonucleotide suchas a CpG oligonucleotide; (13) an immunostimulant and a particle ofmetal salt; and (14) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

As described herein, an antigenic composition may elicit an“immunological response” to an antigen or composition is the developmentin a subject of a humoral and/or a cellular immune response to anantigen present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers broadly to animmune response mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of nonspecific effectercells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular immunogen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. Methodsof measuring cell-mediated immune response include measurement ofintracellular cytokines or cytokine secretion by T-cell populations, orby measurement of epitope specific T-cells.

Accordingly, the term “immunogenic composition” as used herein refersbroadly a composition that comprises an antigenic molecule whereadministration of the composition to a subject results in thedevelopment in the subject of a humoral and/or a cellular immuneresponse to the antigenic molecule of interest. The immunogeniccomposition may be introduced directly into a recipient subject, such asby injection, inhalation, oral, Intranasal and mucosal (e.g.,intra-rectally or intra-vaginally) administration.

For example, the composition may be an antigenic composition or animmunogenic composition. The compositions described herein may compriseat least one excipient, carrier, or adjuvant. Further, the compositionsdescribed herein may be a pharmaceutical composition. In still anotherembodiment, the composition is comprises a pharmaceutical carrier. In afurther embodiment, the composition may elicit an immune response. Inanother embodiment, the immune response may be a protective immuneresponse. In one embodiment, the composition may elicit a humoral immuneresponse, wherein said humoral immune response may be specific for theNPC-1 epitope. In one embodiment, the composition may elicit a cellularimmune response, wherein said cellular immune response may be specificfor the NPC-1 epitope. For example, an antigenic composition comprisingan NPC-1 epitope pepitomimetic may be administered to a mammal whichelicits an immune response including the production of antibodies whichselectively bind the NPC-1 epitope. These antibodies which selectivelybind the NPC-1 epitope may, in turn, act to bind to and trigger theimmunogical clearance (e.g., lysis) of tumor cells expressing an NPC-1epitope.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration through routine experimentation,for example guided by the disclosure herein and the teachings inGoodman, et al. (2011) Goodman & Gilman's The Pharmacological Basis ofTherapeutics [12^(th) Ed.]; Howland, et al. (2005) Lippincott'sIllustrated Reviews: Pharmacology [2^(nd) Ed.]; and Golan, (2008)Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy[2^(nd) Ed.] See, also, Grennaro (2005) [Ed.] Remington: The Science andPractice of Pharmacy [21^(st) Ed.]

Routes of Administration

The compositions described herein may be administered in any of thefollowing routes: buccal, epicutaneous, epidural, infusion, inhalation,intraarterial, intracardial, intracerebroventricular, intradermal,intramuscular, intranasal, intraocular, intraperitoneal, intraspinal,intrathecal, intravenous, oral, parenteral, pulmonary, rectally via anenema or suppository, subcutaneous, subdermal, sublingual, transdermal,and transmucosal. The prcfcrred routes of administration are intravenousinjection or infusion. The administration can be local, where thecomposition is administered directly, close to, in the locality, near,at, about, or in the vicinity of, the site(s) of disease, e.g., tumor,or systemic, wherein the composition is given to the patient and passesthrough the body widely, thereby reaching the site(s) of disease. Localadministration (e.g., injection) may be accomplished by administrationto the cell, tissue, organ, and/or organ system, which encompassesand/or is affected by the disease, and/or where the disease signs and/orsymptoms are active or are likely to occur (e.g., tumor site).Administration can be topical with a local effect, composition isapplied directly where its action is desired (e.g., tumor site).

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms as known in the art. Anybiologically-acceptable dosage form known to persons of ordinary skillin the art, and combinations thereof, are contemplated. Examples of suchdosage forms include, without limitation, chewable tablets, quickdissolve tablets, effervescent tablets, reconstitutable powders,elixirs, liquids, solutions, suspensions, emulsions, tablets,multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules,hard gelatin capsules, caplets, lozenges, chewable lozenges, beads,powders, gum, granules, particles, microparticles, dispersible granules,cachets, douches, suppositories, creams, topicals, inhalants, aerosolinhalants, patches, particle inhalants, implants, depot implants,ingestibles, injectables (including subcutaneous, intramuscular,intravenous, and intradermal), infusions, and combinations thereof.

Other compounds which can be included by admixture are, for example,medically inert ingredients (e.g., solid and liquid diluent), such aslactose, dextrosesaccharose, cellulose, starch or calcium phosphate fortablets or capsules, olive oil or ethyl oleate for soft capsules andwater or vegetable oil for suspensions or emulsions; lubricating agentssuch as silica, talc, stearic acid, magnesium or calcium stearate and/orpolyethylene glycols; gelling agents such as colloidal clays; thickeningagents such as gum tragacanth or sodium alginate, binding agents such asstarches, arabic gums, gelatin, methylcellulose, carboxymethylcelluloseor polyvinylpyrrolidone; disintegrating agents such as starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuff; sweeteners; wetting agents such as lecithin, polysorbates orlaurylsulphates; and other therapeutically acceptable accessoryingredients, such as humectants, preservatives, buffers andantioxidants, which are known additives for such formulations.

Liquid dispersions for oral administration can be syrups, emulsions,solutions, or suspensions. The syrups can contain as a carrier, forexample, saccharose or saccharose with glycerol and/or mannitol and/orsorbitol. The suspensions and the emulsions can contain a carrier, forexample a natural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylccllulosc, or polyvinyl alcohol.

In further embodiments, the present invention provides kits includingone or more containers comprising pharmaceutical dosage units comprisingan effective amount of one or more antibodies and fragments thereof ofthe present invention. Kits may include instructions, directions,labels, marketing information, warnings, or information pamphlets.

Dosages

The amount of NPC-1 epitope peptidomimetics in a therapeutic compositionaccording to any embodiments of this invention may vary according tofactors such as the disease state, age, gender, weight, patient history,risk factors, predisposition to disease, administration route,pre-existing treatment regime (e.g., possible interactions with othermedications), and weight of the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. For example, asingle bolus may be administered, several divided doses may beadministered over time, or the dose may be proportionally reduced orincreased as indicated by the exigencies of therapeutic situation.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of antibodies, and fragmentsthereof, calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the antibodies, and fragmentsthereof, and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an antibodies, andfragments thereof, for the treatment of sensitivity in individuals. Intherapeutic use for treatment of conditions in mammals (e.g., humans)for which the antibodies and fragments thereof of the present inventionor an appropriate pharmaceutical composition thereof are effective, theantibodies and fragments thereof of the present invention may beadministered in an effective amount. The dosages as suitable for thisinvention may be a composition, a pharmaceutical composition or anyother compositions described herein.

The dosage may be administered as a single dose, a double dose, a tripledose, a quadruple dose, and/or a quintuple dose. The dosages may beadministered singularly, simultaneously, and sequentially.

The dosage form may be any form of release known to persons of ordinaryskill in the art. The compositions of the present invention may beformulated to provide immediate release of the active ingredient orsustained or controlled release of the active ingredient. In a sustainedrelease or controlled release preparation, release of the activeingredient may occur at a rate such that blood levels are maintainedwithin a therapeutic range but below toxic levels over an extendedperiod of time (e.g., 4 to 24 hours). The preferred dosage forms includeimmediate release, extended release, pulse release, variable release,controlled release, timed release, sustained release, delayed release,long acting, and combinations thereof, and are known in the art.

It will be appreciated that the pharmacological activity of thecompositions may be monitored using standard pharmacological models thatare known in the art. Furthermore, it will be appreciated that thecompositions comprising a NPC-1 epitope peptidomimetic may beincorporated or encapsulated in a suitable polymer matrix or membranefor site-specific delivery, or may be functionalized with specifictargeting agents capable of effecting site specific delivery. Thesetechniques, as well as other drug delivery techniques are well known inthe art. Determination of optimal dosages for a particular situation iswithin the capabilities of those skilled in the art. See, e.g., Grennaro(2005) [Ed.] Remington: The Science and Practice of Pharmacy [21^(st)Ed.]

Methods of Treatment

The NPC-1 epitope peptidomimetics described herein may be used inmethods for treating cancer, promoting tumor regression, killing tumorcells, activating an immune response against NPC-1 epitope expressingtumor cells (e.g., cytotoxic immune response), activating dendriticcells, or activating antigen-specific immunity comprising administeringan effective amount of a NPC-1 epitope peptidomimetic thereof to asubject in need thereof. Further, the NPC-1 epitope peptidomimeticdescribed herein may be used to manufacture medicaments for use intreating cancer, promoting tumor regression, killing tumor cells,activating an immune response against NPC-1 epitope expressing tumorcells (e.g., cytotoxic immune response), activating dendritic cells, oractivating antigen-specific immunity comprising an effective amount of aNPC-1 epitope peptidomimetic described herein. The NPC-1 epitopepeptidomimetics described herein may be admixed with a pharmaceuticallyacceptable carrier to manufacture a composition for treating cancer,promoting tumor regression, killing tumor cells, activating an immuneresponse against NPC-1 epitope expressing tumor cells (e.g., cytotoxicimmune response), activating dendritic cells, or activatingantigen-specific immunity comprising an effective amount of a NPC-1epitope peptidomimetic described herein.

The cancer treated by the NPC-1 epitope peptidomimetics described hereinmay be lung, breast, pancreas, prostate, uterine, esophageal,colorectal, or liver cancer. The cancer may be a stage 1, 2, 3 or 4cancer. The cancer may have metastasized. The patient may be a mammal,such as a human, suffering from cancer where tumor cells express NPC-1epitopes, aberrant NPC-1 epitopes, and/or tumorigenesis of neoplasticcells expressing a NPC-1 epitope. The amount sufficient to inhibit orreduce the NPC-1 epitope is an amount sufficient to ameliorate thedisorder, which may be monitored as a decrease in either cancerprogression or tumor mass.

The patient may express detectable levels of NPC-1 epitope as detectedin a tumor biopsy sample or in the blood, stool, urine or lymph fluid.See FIG. 1. Further, the patient may be at risk of cancer or a patientwithout symptoms. The methods described herein may be used on cells,e.g., human cells, in vitro or ex vivo. Alternatively, the method may beperformed on cells present in a subject as part of an in vivo (e.g.,therapeutic) protocol.

The NPC-1 epitope peptidomimetics may be admixed with additionalchemotherapeutic agents, cytotoxic agent, antibodies (e.g., NEO-201 orNEO-301 monoclonal antibodies), lymphokine, or hematopoietic growthfactor. The NPC-1 epitope peptidomimetics may also be administered incombination with another antibody, a lymphokine, cytotoxic agent (e.g.,a moiety that inhibits DNA, RNA, or protein synthesis, a radionuclide,or ribosomal inhibiting protein, e.g., ²¹²Bi, ¹³¹I, ¹⁸⁸Re, ⁹⁰Y,vindesine, methotrexate, adriamycin, cisplatin, pokeweed antiviralprotein, Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain,or cytotoxic phospholipase enzyme), immunosuppressive agent (e.g.,cyclosporine, leflunomide, methotrexate, azothiprine, mercaptopurine,dactinomycin, tacrolimus, or sirolimus) or a hematopoietic growthfactor. The NPC-1 epitope peptidomimetics may be label with achemiluminescent label, paramagnetic label (e.g., aluminum, manganese,platinum, oxygen, lanthanum, lutetium, scandium, yttrium, or gallium),an MRI contrast agent, fluorescent label, bioluminescent label, orradioactive label. In the methods described herein, the second agent maybe administered simultaneously or sequentially with the antibody.

The NPC-1 epitope peptidomimetics described herein may be used in themanufacture of compositions for use in treating cancer and methods oftreating cancer including but not limited to solid and soft tumors, suchas esophageal carcinoma, renal cancer, cancer of breast, thyroid,spleen, uterus, kidney, colorectal, lung, prostate, testicles, gastric,pancreas, cervical, bone, skin, brain, head & neck, bladder, head andneck, liver, pancreas, melanoma, osteosarcoma, fibrosarcoma,rhabdomyosarcoma, teratocarcinoma, neuroblastoma, glioma, glioblastomaand hematological malignancies such as acute lymphocytic leukemia,chronic lymphocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, multiple myeloma, Hodgkin's lymphoma andNon-Hodgkin's lymphoma, and wherein the cancer is invasive ormetastatic.

The invention provides for methods of treating a subject with pancreasor colon cancer comprising administering a NPC-1 epitope peptidomimeticsto a subject who may be receiving secondary antihyperplastic therapy.Examples of secondary antihyperplastic therapy include chemotherapy,radiotherapy, immunotherapy, phototherapy, cryotherapy, toxin therapy,hormonal therapy, or surgery. Thus, the invention contemplates use ofthe methods and compositions in conjunction with standard anti-cancertherapies. The patient to be treated may be of any age. One of skill inthe art will recognize the presence and development of other anticancertherapies which may be used in conjugation with the NPC-1 epitopepeptidomimetics.

Determination of dose is within the level of ordinary skill in the art.The NPC-1 epitope peptidomimetics may be administered for acutetreatment, over one week or less, often over a period of one to threedays or may be used in chronic treatment, over several months or years.In general, a therapeutically effective amount of the NPC-1 epitopepeptidomimetics is an amount sufficient to produce a clinicallysignificant change in NPC-1 epitope shed, decreased cancer progression,or decreased tumor size.

Diagnostic Methods

The NPC-1 epitope peptidomimetics may be used in diagnostic methods fordetecting the presence or absence of an NPC-1 epitope. The NPC-1 epitopepeptidomimotics may be used in methods comprising (a) contacting a testsample with an antibody, or fragment thereof, that binds a NPC-1 epitopepeptidomimetic, and (b) assaying for antibody-epitope complexes, whereinthe presence of said epitope is indicative of a carcinoma. Further, theNPC-1 epitope peptidomimetics may be used in a method for detecting thepresence of a NPC-1 epitope in a patient comprising (a) administering tosaid patient a labeled monoclonal antibody, or fragment thereof, thatbinds a NPC-1 epitope peptidomimetic and (b) detecting the presence of aNPC-1 epitope; wherein the presence of said epitope is indicative of acarcinoma. The antibody-epitope complex may be detected by Western blot,radioimmunoassay, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassay, immunoprecipitation assay, precipitation reaction, geldiffusion precipitation reaction, immunodiffusion assay, agglutinationassay, complement-fixation assay, immunohistochemical assay, fluorescentimmunoassay, and protein A immunoassay. The sample may be sample is atissue biopsy, lymph, urine, cerebrospinal fluid, amniotic fluid,inflammatory exudate, blood, serum, stool, or liquid collected from thecolorectal tract.

The NPC-1 epitope peptidomimetics may be used in diagnostic methods fordetecting the presence or absence of an NPC-1 epitope, wherein thepresence of the antigen is indicative of cancer including but notlimited to lung, breast, pancreas, uterine, esophageal, colorectal, orliver cancer. The diagnostic methods may be used with patients at riskof cancer or patients without symptoms.

The antibodies which selectively bind a NPC-1 epitope peptidomimetic maybe recombinant. The fragments of antibodies which selectively bind aNPC-1 epitope peptidomimetics may be a Fab, Fab′, F(ab′)2, Fv, CDR,paratope, or portion of an antibody that is capable of binding theantigen. The antibodies which selectively bind a NPC-1 epitopepeptidomimetics may be chimeric, humanized, anti-idiotypic,single-chain, bifunctional, or co-specific. The antibodies whichselectively bind a NPC-1 epitope peptidomimetics may be or fragment isconjugated to a label, including but not limited to a chemiluminescentlabel, paramagnetic label (e.g., aluminum, manganese, platinum, oxygen,lanthanum, lutetium, scandium, yttrium, or gallium), an MRI contrastagent, fluorescent label, bioluminescent label, or radioactive label.

Additionally, NPC-1 epitopes peptidomimetics may be attached to a solidsupport (e.g., bead, test tube, sheet, culture dish, or test strip) suchas an array.

The method may detect colorectal polyps. The method may further compriseadditional testing for the presence of tumors including but not limitedto benign tumors, malignant tumors, metastatic tumors, andnon-metastatic tumors. For example, the diagnostic method may detectpre-cancerous cells that express a cell marker comprising detecting aNPC-1 epitope.

The method may comprise imaging a NPC-1 epitope by positron emissiontomography (PET), CCD low-light monitoring system, x-ray, CT scanning,scintigraphy, photo acoustic imaging, single photon emission computedtomography (SPECT), magnetic resonance imaging (MRI), ultrasound,paramagnetic imaging, and endoscopic optical coherence tomography.

The invention also provides a method for genetic diagnosis of a risk forcancer comprising taking a nucleic acid sample from a patient, analyzingsaid nucleic acid comprising comparing to cancer specific MUC5ACsequence, wherein if the patient's nucleic acid sample matches thecancer specific MUC5AC sequence, the patient is at risk for developingcancer.

The NPC-1 epitopes may be used as a cancer biomarker. Detection of theNPC-1 epitopes in a biological sample, such as a subject's serum,biopsied neoplastic cells or fecal sample, may be performed by means ofantibodies that selectively bind a NPC-1 epitope peptidomimetic. Forexample, a biological sample (e.g., a tumor, serum or fecal sample) isobtained from a subject, then NPC-1 epitope is measured (e.g., by ELISAor PCR), and compared with corresponding samples from normal subjects.Measuring methods include any method of nucleic acid detection, forexample in situ hybridization using antisense NPC-1 epitope DNA or cRNAoligonucleotide probes, ultra-high throughput sequencing, nanostringtechnology, microarrays, rolling circle amplification,proximity-mediated ligation, PCR, qRT-PCR ChIP, ChIP-qPCR, or NPC-1epitope-binding antibodies. Comparatively high levels of NPC-1 epitopesindicate the presence and/or severity of pancreas or colon cancer, andmay indicate metastasis or poor cancer prognosis.

The NPC-1 epitope pcptidomimetics may be used in SQUID (SuperconductingQuantum Interference Device) techniques for diagnostic methods. TheSQUID technique comprises attaching nanoparticles of iron oxide toantibodies, which are then injected into the patient. If a tumor ispresent, the antibodies with conjugated nanoparticles recognize and bindto the NPC-1 epitope on tumor cells. See, e.g., Hao, et al. (2010)Journal of Physics 43: 474004. In a SQUID method, the patient is thensurrounded with sensitive magnetic coils in a superconducting quantuminterference device (SQUID). A magnetic field is generated and all ofthe metal nanoparticles align in one direction. When the magnetic fieldis broken, the nanoparticles emit an electromagnetic signal as theyrelax back into their original state. By measuring the strength of thesignal, one may tell how many metal particles, and therefore how manytumor cells, may be present, and where in the patient the tumor cellsare located. See, e.g., Shao, et al. (2010) Beilstein Journal ofNanotechnology 1: 142-154.

Samples and Procurement of Samples

The samples used in the methods described herein may be taken from asubject (patient) include but are not limited to a body fluid orsecretion including but not limited to blood, serum, urine, plasma,prostatic fluid, seminal fluid, semen, the external secretions of theskin, respiratory, intestinal, and genitourinary tracts, tears,cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleuralfluid, cyst fluid, secretions of the breast ductal system (and/or lavagethereof), broncho alveolar lavage, lavage of the reproductive system andlavage of any other part of the body or system in the body; samples ofany organ including isolated cell(s) or tissue(s), wherein the cell ortissue can be obtained from an organ selected from, but not limited tolung, colon, ovarian and/or breast tissue; stool or a tissue sample, orany combination thereof. In some embodiments, the term encompassessamples of in vive cell culture constituents. Prior to be subjected tothe diagnostic assay, the sample can optionally be diluted with asuitable diluent.

Numerous well known tissue or fluid collection methods can be utilizedto collect the biological sample from the subject in order to determinethe level of DNA, RNA and/or polypeptide of the marker of interest inthe subject. Examples of tissue or fluid collection methods include, butare not limited to, fine needle biopsy, needle biopsy, core needlebiopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardlessof the procedure employed, once a biopsy/sample is obtained the level ofthe marker may be determined and a diagnosis can thus be made.

Detection of NPC-1 Epitope

The invention provides a method for detecting the NPC-1 epitopes of thisinvention in a biological sample, comprising: contacting a biologicalsample with an antibody specifically recognizing a NPC-1 epitopepeptidomimetic according to the present invention and detecting saidinteraction; wherein the presence of an interaction correlates with thepresence of a NPC-1 epitope in the biological sample.

The NPC-1 epitopes described herein are non-limiting examples of markersfor diagnosing a disease and/or an indicative condition. Each marker ofthe present invention may be used alone or in combination, for varioususes, including but not limited to, prognosis, prediction, screening,early diagnosis, determination of progression, therapy selection andtreatment monitoring of a cancer (e.g., pancreas, liver, colorectal,lung, or breast cancer).

The cancers that may be detected using the methods described hereininclude but are not limited to non-solid and solid tumors, cancer of thebreast, prostate, lung, ovary, colon, uterus, stomach, cervix, liver,pancreas, and wherein the cancer may be invasive or metastatic.

Each NPC-1 epitopes of the present invention may be used alone or incombination, for various uses, including but not limited to, prognosis,prediction, screening, early diagnosis, determination of progression,therapy selection and treatment monitoring of cancers such as non-solidand solid tumors, cancer of the breast, prostate, lung, ovary, colon,uterus, stomach, cervix, liver, pancreas, and wherein the cancer may beinvasive or metastatic. Such a combination may optionally comprise anysubcombination of markers, and/or a combination featuring at least oneother marker, for example a known marker. Furthermore, such acombination may optionally and preferably be used as described abovewith regard to determining a ratio between a quantitative orsemi-quantitative measurement of any marker described herein to anyother marker described herein, and/or any other known marker, and/or anyother marker.

Markers of the present invention may optionally be used alone or incombination with known markers for lung cancer, including but notlimited to CEA, CA15-3, 3-2-microglobulin, CA19-9, TPA, and/or incombination with the known proteins for the variant marker as describedherein.′

Markers of the present invention might optionally be used alone or incombination with known markers for ovarian cancer, including but notlimited to CEA, CA125 (Mucin 16), CA72-4TAG, CA-50, CA 54-61, CA-195 andCA 19-9 in combination with CA-125, and/or in combination with the knownproteins for the variant marker as described herein.

Markers of the present invention might optionally be used alone or incombination with known markers for colon cancer, including but notlimited to CEA, CA19-9, CA50, and/or in combination with the knownproteins for the variant marker as described herein.

Typically the level of the marker in a biological sample obtained fromthe subject is different (i.e., increased or decreased) from the levelof the same marker in a similar sample obtained from a healthyindividual (examples of biological samples are described herein).

Determining the level of the same marker in normal tissues of the sameorigin may be effected along-side to detect an elevated expressionand/or amplification and/or a decreased expression, of the marker asopposed to the normal tissues.

The present invention also provides methods, uses, devices and assaysfor the diagnosis of cancers such as non-solid and solid tumors, cancerof the breast, prostate, lung, ovary, colon, uterus, stomach, cervix,liver, pancreas, and wherein the cancer may be invasive or metastatic.Optionally a plurality of markers may be used with the presentinvention. The plurality of markers may optionally include a markersdescribed herein, and/or one or more known markers. The plurality ofmarkers is preferably then correlated with the disease or condition. Forexample, such correlation may optionally comprise determining theconcentration of each of the plurality of markers, and individuallycomparing each marker concentration to a threshold level. Optionally, ifthe marker concentration is above or below the threshold level(depending upon the marker and/or the diagnostic test being performed),the marker concentration correlates with the disease or condition.Optionally and preferably, a plurality of marker concentrationscorrelates with the disease or condition.

Alternatively, such correlating may optionally comprise determining theconcentration of each of the plurality of markers, calculating a singleindex value based on the concentration of each of the plurality ofmarkers, and comparing the index value to a threshold level. Also, suchcorrelating may optionally comprise determining a temporal change in atleast one of the markers, and wherein the temporal change is used in thecorrelating step.

Such correlating may optionally comprise determining whether at least“X” number of the plurality of markers has a concentration outside of apredetermined range and/or above or below a threshold (as describedabove). The value of “X” may optionally be one marker, a plurality ofmarkers or all of the markers; alternatively or additionally, ratherthan including any marker in the count for “X”, one or more specificmarkers of the plurality of markers may optionally be required tocorrelate with the disease or condition (according to a range and/orthreshold).

Correlating may optionally comprise determining whether a ratio ofmarker concentrations for two markers is outside a range and/or above orbelow a threshold. Optionally, if the ratio is above or below thethreshold level and/or outside a range, the ratio correlates with thedisease or condition. Optionally, a combination of two or more thesecorrelations may be used with a single panel and/or for correlatingbetween a plurality of panels. Optionally, the method distinguishes adisease or condition with a sensitivity of at least 70% at a specificityof at least 85% when compared to normal subjects. As used herein,sensitivity relates to the number of positive (diseased) samplesdetected out of the total number of positive samples present;specificity relates to the number of true negative (non-diseased)samples detected out of the total number of negative samples present.Preferably, the method distinguishes a disease or condition with asensitivity of at least 80% at a specificity of at least 90% whencompared to normal subjects. More preferably, the method distinguishes adisease or condition with a sensitivity of at least 90% at a specificityof at least 90% when compared to normal subjects. Also more preferably,the method distinguishes a disease or condition with a sensitivity of atleast 70% at a specificity of at least 85% when compared to subjectsexhibiting symptoms that mimic disease or condition symptoms.

A marker panel may be analyzed in a number of fashions well known tothose of skill in the art. For example, each member of a panel may becompared to a “normal” value, or a value indicating a particularoutcome. A particular diagnosis/prognosis may depend upon the comparisonof each marker to this value; alternatively, if only a subset of markersis outside of a normal range, this subset may be indicative of aparticular diagnosis/prognosis. The skilled artisan will also understandthat diagnostic markers, differential diagnostic markers, prognosticmarkers, time of onset markers, disease or condition differentiatingmarkers, may be combined in a single assay or device. Markers may alsobe commonly used for multiple purposes by, for examplc, applying adifferent threshold or a different weighting factor to the marker forthe different purpose(s).

The panels may comprise markers for the following purposes: diagnosis ofa disease; diagnosis of disease and indication if the disease is in anacute phase and/or if an acute attack of the disease has occurred;diagnosis of disease and indication if the disease is in a non-acutephase and/or if a non-acute attack of the disease has occurred;indication whether a combination of acute and non-acute phases orattacks has occurred; diagnosis of a disease and prognosis of asubsequent adverse outcome; diagnosis of a disease and prognosis of asubsequent acute or non-acute phase or attack; disease progression (forexample for cancer, such progression may include for example occurrenceor recurrence of metastasis).

The above diagnoses may also optionally include differential diagnosisof the disease to distinguish it from other diseases, including thosecancers such as non-solid and solid tumors, cancer of the breast,prostate, lung, ovary, colon, uterus, stomach, cervix, liver, pancreas,and wherein the cancer may be invasive or metastatic that may featureone or more similar or identical symptoms.

One or more diagnostic or prognostic indicators are correlated to acondition or disease by merely the presence or absence of theindicator(s). In other embodiments, threshold level(s) of a diagnosticor prognostic indicator(s) can be established, and the level of theindicator(s) in a patient sample can simply be compared to the thresholdlevel(s). The sensitivity and specificity of a diagnostic and/orprognostic test depends on more than just the analytical “quality” ofthe test—they also depend on the definition of what constitutes anabnormal result. In practice, Receiver Operating Characteristic curves,or “ROC” curves, are typically calculated by plotting the value of avariable versus its relative frequency in “normal” and “disease”populations, and/or by comparison of results from a subject before,during and/or after treatment.

NPC-1 epitopes may be featured as a biomarker for detecting cancers suchas non-solid and solid tumors, cancer of the breast, prostate, lung,ovary, colon, uterus, stomach, cervix, liver, pancreas, and wherein thecancer may be invasive or metastatic.

The present invention optionally and preferably encompasses any aminoacid sequence or fragment thereof encoded by a nucleic acid sequencecorresponding to NPC-1 epitopes as described herein. Any oligopeptide orpeptide relating to such an amino acid sequence or fragment thereof mayoptionally also (additionally or alternatively) be used as a biomarker.

The present invention provides a method for detecting a polynucleotideof this invention in a biological sample, using NAT based assays,comprising: hybridizing the isolated nucleic acid molecules oroligonucleotide fragments of at least about a minimum length to anucleic acid material of a biological sample and detecting ahybridization complex; wherein the presence of a hybridization complexcorrelates with the presence of the polynucleotide in the biologicalsample. Non-limiting examples of methods or assays are described herein.The present invention also relates to kits based upon such diagnosticmethods or assays.

Additionally, the NPC-1 epitopes may be used as specific biomarkers forpancreas and colon cancer, and can be measured in biopsied tissue aswell as in subject serum and fecal samples, as described herein.Additionally, diagnostic procedures used to detect colorectal cancerincluding but not limited to fecal occult blood test (FOBT),colonoscopy, computed tomographic colonography (virtual colonoscopy)[detects colorectal lesions larger than 6 mm in diameter with the samesensitivity as colonoscopy], flexible sigmoidoscopy, double-contrastbarium enema, and digital rectal examination. Winawer, et al. (1997) AmJ. Gastroenterology 112: 594-642; Blum (1995) Eur. J. Canc. 31: 1369-72;Ransohoff & Sandler (2002) N. Engl. J. Med. 346: 346-44; Bruzzi (2002)N. Engl. J. Med. 346: 1672-74; and Laghi, et al. (2002) Am. J. Surg.183: 124-31.

Immunoassays

The NPC-1 peptidomimetics may be used in immunoassays to qualitativelyor quantitatively detect and analyze markers in a sample. This methodcomprises providing an antibody specifically binds to a NPC-1 epitopepeptidomimetic; contacting a sample with the antibody; and detecting thepresence of a complex of the antibody bound to the marker in the sample.

An NPC-1 epitope may be detected and/or quantified using any of a numberof well recognized immunological binding assays. Useful assays include,for example, an enzyme immune assay (EIA) such as enzyme-linkedimmunosorbent assay (ELISA), a radioimmunoassay (RIA), a Western blotassay, or a slot blot assay. See, e.g., U.S. Pat. Nos. 4,366,241;4,376,110; 4,517,288; and 4,837,168. Generally, a sample obtained from asubject can be contacted with the antibody specifically binds the NPC-1epitope.

Optionally, the antibody can be fixed to a solid support to facilitatewashing and subsequent isolation of the complex, prior to contacting theantibody with a sample. Examples of solid supports include but are notlimited to glass or plastic in the form of, e.g., a microtiter plate, astick, a bead, or a microbead. Antibodies may be attached to a solidsupport.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed may be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.Alternatively, the marker in the sample can be detected using anindirect assay, wherein, for example, a second, labeled antibody is usedto detect bound marker-specific antibody, and/or in a competition orinhibition assay wherein, for example, a monoclonal antibody which bindsto a distinct epitope of the marker are incubated simultaneously withthe mixture.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations. Usually the assays will becarried out at ambient temperature, although they can be conducted overa range of temperatures (e.g., about 10C-40° C.).

The immunoassay can be used to determine a test amount of a marker in asample from a subject. First, a test amount of a marker in a sample maybe detected using the immunoassay methods described above. If a markeris present in the sample, it will form an antibody-marker complex withan antibody specifically binds the marker under suitable incubationconditions described above. The amount of an antibody-marker complex canoptionally be determined by comparing to a standard. As noted above, thetest amount of marker need not be measured in absolute units, as long asthe unit of measurement can be compared to a control amount and/orsignal. Several immunoassays are known in the art and the NPC-1 epitope,and antibodies specific for said antigens described herein may used insuch immunoassays including but not limited to radio-immunoassay (RIA),enzyme linked immunosorbent assay (ELISA), magnetic immunoassay,immunoblot, Western blot, immunoprecipitation assays,immunohistochemical analysis, and fluorescence activated cell sorting(FACS). See Wild, (2008) [Ed.] The Immunoassay Handbook [3^(rd) Ed.]Elsevier.

Radio-Imaging Methods

The NPC-1 epitope peptidomimetics and antibodies that selectively bindNPC-1 epitope peptidomimetics may be used in radio-imaging methods todiagnosis cancer including pancreatic and colorectal cancer, or monitorthe progression of tumors. These methods include but are not limited to,positron emission tomography (PET) single photon emission computedtomography (SPECT). Both of these techniques are non-invasive, and canbe used to detect and/or measure a wide variety of tissue events and/orfunctions, such as detecting cancerous cells for example. SPECT mayoptionally be used with two labels simultaneously. See U.S. Pat. No.6,696,686.

Commercial Applications and Methods

The present invention further provides for the production of NPC-1epitope, antibodies and antigen binding fragments thereof whichselectively bind to NPC-1 epitope to reach commercial quantities. TheNPC-1 epitope, antibodies and antigen binding fragments thereof whichselectively bind to NPC-1 epitope may be produced on a large scale,stored if necessary, and supplied to hospitals, clinicians or otherhealthcare facilities.

Methods of production, storage, and distribution of NPC-1 epitope,antibodies and antigen binding fragments thereof which selectively bindto NPC-1 epitope may be produced by the methods disclosed herein.Following production, the NPC-1 epitope, antibodies and antigen bindingfragments thereof which selectively bind to NPC-1 epitope may beharvested, purified, and optionally stored prior to a patient'streatment. For example, once a patient presents with an indication suchas, for example, pancreatic, colorectal, esophageal, oral, or breastcancer, NPC-1 epitope, antibodies and antigen binding fragments thereofwhich selectively bind to NPC-1 epitope may be ordered and provided in atimely manner. Accordingly, the present invention relates to methods ofproducing NPC-1 epitope to attain antibodies on a commercial scale,pharmaceutical compositions comprising antibodies and antigen bindingfragments thereof which selectively bind to NPC-1 epitope, as well asmethods of providing (i.e., producing, optionally storing, and selling)antibodies and antigen binding fragments thereof which selectively bindto NPC-1 epitope to hospitals and clinicians. The production of NPC-1epitope, antibodies and antigen binding fragments thereof whichselectively bind to NPC-1 epitope may be scaled up for commercial use.

The present invention also provides for methods of conducting apharmaceutical business comprising establishing a distribution systemfor distributing the preparation for sale or may include establishing asales group for marketing the pharmaceutical preparation.

All publications (e.g., Non-Patent Literature), patents, patentapplication publications, and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All such publications (e.g.,Non-Patent Literature), patents, patent application publications, andpatent applications are herein incorporated by reference to the sameextent as if each individual publication, patent, patent applicationpublication, or patent application was specifically and individuallyindicated to be incorporated by reference.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which arm includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Identification of the NPC-1 Epitope

A NEO-100 antibody was generated in mice immunized with the so-called“Hollinshead colon cancer vaccine”. Hollinshead, et al. (1985) Cancer56: 480-489. A NEO-100 antibody and the chimeric form, NEO-101, aredescribed in U.S. Pat. Nos. 7,314,622 and 7,763,720. Several proteinpurifications were prepared using both mouse NPC-1 and recombinant,chimeric NEO-101 antibodies. Tumor cell lines including LS174T and HT-29(human colorectal tumor), CFPAC-1 (human pancreatic tumor), colon cancerpatient tumor specimen, and the Hollinshead colon cancer vaccines servedas tumor antigen sources for protein extracts. The NPC-1 epitope issecreted into the medium of the human tumor cell lines, and the antigenwas purified from tumor cell supernatant of cells grown in the absenceof serum for 5 to 7 days. NEO-101 antibody was coupled to resins for theantigen purification, including magnetic beads, for simple adsorption,washing, and elution from the beads. Protein that eluted from a NEO-100antibody-beads was studied further for antigen presence,characterization, and identification.

Western blotting of human tumor cell extracts and supernatants usingNEO-101 antibody. Proteins from AsPC-1, LS174T or CFPAC-1 cellsupernatants or cell pellet detergent extracts were run on SDS-PAGE,transferred to PVDF membrane, then stained with NEO-101 antibody. A highmolecular mass species cross-reactive with a NEO-100 antibody estimatedto be 1,000-2,000 kDa was identified by SDS-PAGE. A protein immunoblotof tumor antigen from cells using NEO-101 antibody including AsPC-1 cellpellet, AsPC-1 supernatant, LS174T cell pellet, LS174T supernatant,CFPAC-1 cell pellet, and CFPAC-1 supernatant along with molecular weightmarkers.

Immunopurified protein from LS174T tumor cells was subjected toproteolytic cleavage by either trypsin or protease V8, followed byWestern Blot analysis of the protein fragments. A 1,000-2,000 kDaimmunopurified antigen was digested into four discrete fragments rangingin mass from approximately 60 kDa to 220 kDa, each containing an NPC-1immunoreactive peptide epitope. A protein immunoblot of proteolyticdigested tumor antigen from cells using NEO-101 antibody was run withLS174T cell pellet, LS174T supernatant, trypsin-digested immunopurifiedantigen, protease V8-digested immunopurified antigen along with amolecular weight marker. The data suggested that there are multipleNEO-101 antibody binding regions present on each molecule of the tumorantigen.

The NPC-1 epitope was prepared for identification by mass spectrometryby running immunopurified antigen preparation from several differenttumor sources on SDS-PAGE, excising the high molecular mass, NPC-1immunoreactive band from the polyacrylamide gel, and subjecting theprotein to trypsin digestion followed by LC/MS/MS on an LTQ Orbitrap XLmass spectrometer. Trypsin peptide product ion data defined by mass andcharge were searched against the concatenated forward and reverse IPIhuman database using the Mascot search engine. The database was appendedwith commonly observed background proteins to prevent false assignmentsof peptides derived from those proteins. Mascot output files were parsedinto the Scaffold program for filtering to assess false discovery ratesand allow only correct protein identifications. Rat, mouse, and humanderived samples are searched against the International Protein Index(IPI) database. The antigen sources for these experiments were derivedfrom human colorectal (LS174T, HT-29) and pancreatic (CFPAC-1) tumorcell lines. The results of six mass spectrometry experiments suggestedthe presence of MUC5AC-derived peptides in the NPC-1 immunopurificationpreparation.

The amino acid sequence of MUC5AC as reported in the IPI database(IPI00103397). The sequence consists of 5,030 amino acids with apredicted mass of 526,585 Da (without post-translational modificationsincluding glycosylation). Comparing the peptide coverage from the massspectrometetry experiments with the amino acid sequence of MUC5AC (SEQID NO: 36), and other peptide coverage maps, reveal that most of thepeptides sequenced after trypsin digestion derive from either theN-terminal third or the C-terminal third of the molecule. In eachexperiment, there were very few peptides that derived from the centralthird of the MUC5AC molecule, which contains “tandem repeats” of 8 aminoacid residues including, for example, TTSTTSAP (SEQ ID NO: 42), GSTPSPVP(SEQ ID NO: 43), and TASTTSGP (SEQ ID NO: 44). Silverman, et al., (2001)Glycobiology 11: 459-71. This region of MUC5AC is highly glycosylated innormal MUC5AC-expressing tissues, such as lung and colon endothelium. Itis probable that the lack of peptide sequence coverage in the centralregion of MUC5AC, as detected by mass spectrometry, is due to a highdegree of glycosylation in the region, which interferes with digestionby trypsin. These results suggest that the tandem repeat region ofMUC5AC comprises at least one NPC-1 epitope.

Example 2 NPC-1 Epitope Knockdown

A small inhibitory RNA (siRNA) target sequence designed against a regionof MUC5AC was used in cells known to express the NPC-1 epitope. SeveralsiRNA oligonucleotides were designed based upon MUC5AC sequences. Thesequences of the human MUC5AC siRNA oligonucleotides are shown in Table5:

TABLE 5 Sequence of MUC5AC siRNA oligonucleotides Oligonucleotide StrandSequence siRNA ID #: s9074 Sense AGAUGUGCCUCAACUACGAtt (SEQ ID NO: 51)Anti- UCGUAGUUGAGGCACAUCUtg Sense (SEQ ID NO: 52) siRNA ID #: s9075Sense GCUCUGGAACGUGAGCAUAtt (SEQ ID NO: 53) Anti- UAUGCUCACGUUCCAGAGCcgSense (SEQ ID NO: 54) siRNA ID #: s9076 Sense GCGUGCUCGUCGACAACUAtt(SEQ ID NO: 55) Anti- UAGUUGUCGACGAGCACGCgg Sense (SEQ ID NO: 56)

The siRNAs were transfected into LS174 and CFPAC-1 tumor cells, as wellas A549 cells. A549 is a human lung adenocarcinoma cell line thatexpresses MUC5AC (as shown by PCR and detection using commerciallyavailable antibodies against MUC5AC), but not NPC-1 epitope. The MUC5ACspecies expressed by A549 cells is not immunoreactive with NEO-101antibody, a characteristic that demonstrates the tumor specificity ofthe NPC-1 epitope in contrast to the commercially available antibodiesagainst MUC5AC. The A549 cells serve as a control to show thepancreas/colon tumor-binding specificity of the NEO-101 antibody.Following transfection of the siRNA into tumor cells, the MUC5ACexpressed by the cells was measured by specific PCR to measure thelevels of MUC5AC mRNA, and a sandwich ELISA using NEO-101 antibody tomeasure the levels of MUC5AC. The data shown in Table 6 demonstrate thata cocktail of three siRNA oligonucleotides resulted in significantdecreases of both MUC5AC mRNA and NEO-101-reactive MUC5AC protein (e.g.,NPC-1 epitope).

TABLE 6 siRNA knockdown of MUC5AC in human pancreatic (CFPAC-1) andcolorectal (LS174T) tumor cells Normalized Normalized MUC5AC MUC5ACmRNA* protein** Untreated CFPAC-1 1 1 CFPAC-1 Treated with  20 pmol0.2737 0.6967 SiRNA cocktail  50 pmol 0.3057 0.6901 200 pmol 0.13680.3566 Untreated LS174T 1 1 LS174T Treated with  20 pmol 0.6917 0.53SiRNA cocktail  50 pmol 0.3858 0.402 200 pmol 0.235 0.117 *MUC5AC mRNAlevel was measured by RT-PCR, normalized as a percent of mRNA levelsdetected in untreated cells. **MUC5AC protein level secreted into cellsupernatants was measured by sandwich ELISA [NEO-101 to capture andanti-MUC5AC antibody 1-13M (Abcam catalog #ab24070) to detect],normalized as a percent detected in untreated cells.

The amount of decreased MUC5AC expression was dependent on the dose ofthe siRNA cocktail transfected into the cells. Approximately 70%-90% ofMUC5AC expression (mRNA and protein) was inhibited in both LS174T andCFPAC-1 cell lines at 200 pmoles of the siRNA cocktail. These resultsconfirmed that MUC5AC is the target of the NEO-101 antibody. The A549cells used as a control in these experiments showed decreased MUC5ACexpression by mRNA analysis but there was no change in the NEO-101sandwich ELISA because the MUC5AC expressed by these cells is notrecognized by a NEO-100 antibody. Thus, the siRNA data demonstrates thatreducing MUC5AC expression lead to a concurrent decrease in NEO-101antibody binding.

Example 3 NPC-1 Epitope Mapping

The data by western blots of trypsin-digested MUC5AC indicated thatthere may be several NEO-101 antibody binding sites on each molecule ofMUC5AC, suggesting that a binding region may be present in each of thetandem repeat units located in the central region of the molecule. Anexemplary amino acid sequence of MUC5AC is presented in SEQ ID NO: 36with an exemplary nucleic acid sequence encoding MUC5AC presented in SEQID NO: 37. Therefore, a recombinant expression plasmid was designed andconstructed that encoded residues upstream of tandem repeat units andtwo tandem repeat units (“MUC5AC-long” SEQ ID NO: 38 with thecorresponding nucleic acid sequence of SEQ ID NO: 39). The MUC5AC longpeptide corresponds to amino acid residues 2636 to 2942 of the MUC5ACprotein (SEQ ID NO: 36). A second expression plasmid was designed andconstructed that encoded primarily a short domain which connects to thecentral repetitive region and only a portion of the tandem repeatresidues (“MUC5AC-short” SEQ ID NO: 40 with the nucleic acid encoding itin SEQ ID NO: 41). The MUC5AC short peptide corresponds to amino acidresidues 2636 to 2763 of the MUC5AC protein (SEQ ID NO: 36). Thesesmaller fragments of the large MUC5AC molecule were predicted tocomprise NEO-101 antibody binding regions that contain the NPC-1epitope(s).

The DNA sequences that contained NEO-101 antibody binding regions, basedon the amino acid sequence, were back-translated to nucleic acidsequence and the DNAs were synthesized by methods well-known in the art.These DNA fragments were cloned into a mammalian expression plasmid bystandard techniques, and several independent clones were transfectedinto Chinese Hamster Ovary (CHO) cells that were shown previously not toexpress the NPC-1 epitope. Analysis of the CHO cells following thetransfection demonstrated immunoreactivity with a NEO-100 antibody inseveral of the plasmid clones. Experiments were performed to testbinding by immunofluorescence and immunoprotein blotting of extractsfrom transfected cells.

Immunofluorescence quantitation data showed that NEO-101 antibody boundto 11%-80% of CHO cells transfected with different plasmid clones of theMUC5AC short construct, and 76%-88% of CHO cells transfected withdifferent plasmid clones of the MUC5AC long construct. Western blotanalysis using NEO-101 to probe CHO cell extracts following transfectionwith the MUC5AC short and MUC5AC long plasmid clones was performed. TheNEO-101 antibody binding region was expressed by both of theseMUC5AC-related-peptides as confirmed by Western blot analysis. Themolecular mass of the immunoreactive protein bands represents thepredicted mass of the protein fragment, including glycosylation thatoccurs in the mammalian CHO cells. The data further confirm that theNPC-1 antigen (e.g., a NEO-100 antibody binding region) is contained inthe MUC5AC-related fragments isolated and expressed in the transfectedcells.

The results of these experiments show that at least one NPC-1 epitope iscontained within both the 307 amino acid-fragment of the MUC5AC longpeptide and within the 128 amino acid-fragment of the MUC5AC shortpeptide. These results also suggest that the NPC-1 epitope may be aconformational epitope rather than a liner epitope.

Example 4 Deletion Constructs for Detailed Epitope Mapping of the MUC5ACLong Antigen

The NEO-101 binding region was shown to be expressed by the MUC5AC,successive truncations starting at either the N-terminus or theC-terminus of the construct may be generated by standard molecularbiology techniques to identify a region which is involved in NEO-101antibody binding to MUC5AC. Six truncation constructs were maderepresenting C-terminal truncations of the full-length MUC5AC protein(SEQ ID NO: 36).

TABLE 7 Truncation constructs of MUC5AC Construct Modification SEQ ID NOBinds NPC-1? 1-338 C-terminal truncation 50 Yes 1-289 C-terminaltruncation 49 Yes 1-187 C-terminal truncation 48 Yes 1-151 C-terminaltruncation 47 Yes 1-136 C-terminal truncation 46 No 1-85 C-terminaltruncation 45 No

293T cells were cultured on FBS coated cover slips for 24 hours. Cellswere transiently transfected with 2 μg of MUC5AC constructs of Table 8with LIPOFECTAMINE®. Cells were grown for about 72 hours, fixed withabout 4% PFA in PBS, washed with PBS, permeabilized with about 0.2%triton X-20 in PBS and washed with PBS. Cells were then blocked withabout 1% BSA in PBS, and about 2 μg/ml NEO-101 antibody was added tosample wells and about 2 μg/ml of an Isotype control was added for 1hour at room temp. Cells were washed with PBS and second anti human-FITCantibody (1:500) was added to all wells and to the second antibodycontrol wells. Cells were washed and mounted on slides with DAPI hardmount. Cover slides were allowed to sit over night at about 4° C. Slideswere visualized with a Nikon Eclipse Ti microscope with an Andor camera.At least 3 random fields were counted per transfection.

This strategy was used to identify an about 15 amino acid-region thatcontains a NPC-1 epitope: GCPVTSTPVTAPSTP (SEQ ID NO: 35). The MUC5ACconstructs with 338, 289, 187, and 151 amino acid residues all hadbinding activity with the NEO-101 antibody. See TABLE 2B. The deletions151 and 85 showed no binding. The second antibody and the IgG controlswere also negative for binding to the 293T cell lines transfected withthe MUC5AC 338 residue construct (SEQ ID NO: 50). These data suggestthat the GCPVTSTPVTAPSTP (SEQ ID NO: 35) sequence is involved in thebinding of the NPC-101 antibody to MUC5AC. This is a repetitive sequencethat also has some changes in it which are also probable scaffold forthe binding sites for the NEO-101 antibody. This repetitive sequence andvariations can be found in the longer deletions of MUC5AC and MUC5ACitself.

Example 5 NPC-1 Epitope is a Specific Biomarker for Pancreatic andColorectal Cancer

The NEO-101 antibody is specific for MUC5AC-related antigen expressedfrom human colon (LS174T) and pancreas (CFPAC-1) cancer cell lines.MUC5AC-related antigen expressed by these two cancer cell linescomprised the NPC-1 epitope, and competed effectively with native MUC5ACantigen previously coated on ELISA plates for binding to NEO-101 in theassay. As a control, non-NPC-1-bearing MUC5AC expressed by A549 lungadenocarcinoma cells did not compete with NEO-101 antibody binding tonative MUC5AC coated to the ELISA plates.

LS174T and CFPAC-1 cells were grown on cover slips coated in FBS for 48hours. Cells were then fixed with 4% PFA in PBS, washed with PBS,permeabilized with 0.2% Triton X-100, washed with PBS and blocked with1% BSA in PBS. Cells were incubated with either 2 μg/ml NEO-101, MS-X,2-11M1, H160, 351-450, 2-12M1, or 1-13M1. Cells were washed with PBS andthen second antibody was added anti-human-FITC, anti-mouse-FITC oranti-rabbit-FITC (1:500), cells were washed and mounted on slides withDAPI hard mount. Cover slides were allowed to sit over night at 4° C.Slides were visualized with a Nikon Eclipse Ti microscope with an Andorcamera.

All of the antibodies stained the LS174T cells. The staining patternsand localization looked about the same for all antibodies tested. Therewere differences in cell staining on the CFPAC-1 cells compared to theLS174T cells. NEO-101 stained about 50% of the cells. MS-X and 2-11M1stained less than 5% of the cells. 351-450 and 2-12M1 did not have anystaining with the CFPAC-1 cells. H160 and 1-13M1 stained 100% of theCFPAC-1 cells.

This data suggests that all the antibodies can detect the colorectalMUC5AC protein with the same efficiency but in pancreatic cancer cellsthere are variations in the staining patterns between different MUC5ACantibodies. This suggests that NEO-101 may detect both types of MUC5ACand that other commercial antibodies do not recognize the same epitope.

A homologous ELISA assay (adapted from an ImmunoBooster® ELISA kit,Bioworld Consulting Laboratories, LLC, Mt. Airy, Md.) was designed usingNEO-101 antibody as both capture and detection reagent (e.g., a sandwichELISA was developed using NEO-101 antibody as the capture reagent usinga biotin-labeled NEO-101 used as the detection antibody.) Thishomologous antibody format was possible due to the discovery of multipleNPC-1 antigen binding sites expressed by the cancer-associatedMUC5AC-related antigen. Serum samples were procured from variouscommercial and private sources. The assay developed here used serum fromcolorectal and pancreatic cancer patients, and serum from healthy blooddonors.

Microtiter plates (96-well Nunc Maxisorp) were coated with purifiedunlabeled NEO-101 antibody at about 10 μg/mL in 0.5 M sodium carbonatepH 9.5 overnight at about 25° C. Plates were then blocked with 1% skimmilk made in Tris-Buffered Saline (TBS) containing 5 mM EDTA and 1%sucrose for about 4 hours at about 2° C. Plates prepared in this mannermay be stored dried and sealed for at least about 8 months. Alldilutions were made in ImmunoBooster® buffers (Bioworld ConsultingLaboratories, LLC) supplemented with 20 mM EDTA. Wash buffer was TBScontaining 0.05% Tween®-20 non-ionic detergent. A detergent extract ofcultured human LS174T colorectal tumor cells was used as a source ofNPC-1 antigen to derive a standard curve. Extracts derived from humanpancreatic CFPAC-1 tumor cells or human lung A549 tumor cells weregenerated similarly. Tumor cell lines were purchased from American TypeCulture Collection (Manassas, Va.) and grown in RPMI medium containing10% FBS (heat-inactivated) with 8 mM glutamine. To measure directbinding of NEO-101 to the MUC5AC-related antigen, CFPAC-1 cells weregrown in serum-free medium for about 5 days and the conditioned mediumwas filtered and stored in large one large lot at about 4° C.

The sandwich ELISAs were performed by diluting the cell extract standardon each plate, next to patient or normal serum samples diluted 1:24 inthe diluent. All incubations were performed at about 25° C. and allvolumes were about 100 μl per well. The plates were incubated for 15 minand washed three times with wash buffer. The biotin-labeled NEO-101 wasthen added to the wells at 1 g/mL, incubated for about 15 min, andplates were washed three times. Peroxidase-conjugated streptavidin(1:5,000 dilution) was added to the plates for about 15 min, and plateswere washed three times with wash buffer and two times with TBS. Theassay was developed by the addition of TMB substrate (BioFX LaboratoriesInc.) to the plates, incubation for about 15 minutes, then the colorreaction was stopped with the addition of 0.5 M sulfuric acid. The datawas acquired by measuring absorbance at 450 nm. The data collected wasprocessed using GraphPad Prism or Microsoft Excel software packages.

This ELISA was used to evaluate the serum of colorectal and pancreaticcancer patients (n=42), serum from healthy blood donors (n=75), andserum from potentially interfering disease states such as asthma,chronic obstructive pulmonary disease, irritable bowel syndrome andCrohn's disease (n=56). Analysis of these various serum samplesdemonstrates the use of the NPC-1 antigen biomarker assay to detectNPC-1 epitope (e.g., aberrant MUC5AC) shed into the blood of colorectalcancer patients. An NPC-1 ELISA test may detect aberrant MUC5AC fromcolon cancer patients. C1 and C2 are normal serum samples from healthyblood donors. AB pool is serum pooled from many healthy blood donors.All other samples numbered #1 through #17 are serum collected from coloncancer patients. The use of NEO-101 antibody as the coating antibody(capture antibody) and biotin-labeled NEO-101 as the detection antibodyis highly specific, and may be explained by the presence of multiplebinding regions (i.e., epitopes) on the same antigen molecule, such thatsteric hindrance is obviated.

Patients with colorectal or pancreas cancer were asked to participate ina study. Serum samples were received and stored at about −35° C. untilthe time of testing. Tumor biopsy slides were received at ambient roomtemperature and subsequently analyzed by IHC using biotin-labeledantibodies. Patient information was also provided, containing limitedclinical data for the patient sample (disease stage, currentmedications). For each patient enrolled, 1, 2 or 3 serum samples wereprovided for each patient separated by approximately 1-month. A group of“normal healthy” serum samples was included for comparison. These werepurchased from a large metropolitan blood bank and comprised a group ofself-proclaimed normal individual males and females of mixed race aged40-to 59-years-old. The actual health status of these donors is unknown,thus comparison of any sample to this normal donor group may be donewith appropriate caution.

The NEO-101 serum ELISA was performed using a standard antigen preparedfrom a cultured cell line extract from tumor cells known to express theNPC-1 antigen. Triplicates of a 1/24 dilution of serum samples fromgroups of “healthy normal” donors and clinically diagnosed colon andpancreas cancer patients were tested in the assay and the raw data wereinterpolated from the standard curve. Expression of the NPC-1 antigenwas determined relative to this standard antigen preparation(equivalents of LS174T cells/well).

Results showed that interfering disease states, which are expectedtypically to have elevated serum MUC5AC, did not express higher levelsof NPC-1 epitope compared to controls. Further, comparison of the serumMUC5AC levels from colorectal and pancreatic cancer patients with serumfrom healthy controls demonstrated the assay's ability to differentiatethe cancer patients from the normal donors with approximately 0.7 logunits difference. Moreover, the NEO-101 ELISA accurately differentiatedpatients with active or metastatic disease from patients who had noevidence of disease. Notably, in a side-by-side comparison of theNEO-101 ELISA to a commercial ELISA for CA19-9, the NEO-101 ELISA provedsuperior.

Patients enrolled on the clinical diagnostic study agreed to providetheir tumor biopsy or surgical specimen to be stainedimmunohistochemically with NEO-101. Tumor sections were prepared asslides, and two additional slides were prepared for negative control(human IgG1) and positive control (cytokeratin) staining to ensurequality controls for the IHC method. More specifically, tumor biopsyspecimens from colorectal and pancreas cancer patients weredeparaffinized at about 60° C. for 30 min prior to staining withNEO-101. Subsequently, all staining steps were carried out at about 25°C. Slides (4 μm) were blocked with Peroxo-Bloc® inhibitor (ZymedLaboratories) for about 2 min, rinsed with phosphate-buffered saline(PBS), and blocked with CAS (Zymed) for an additional about 10 minutes.Slides were stained with about 10 μg/mL of biotin-labeled NEO-101 for 1hour, and washed three times with PBS containing 0.05% Tween®-20non-ionic detergent. Previous titration of biotinylated-NEO-101demonstrated about 10 μg/mL to be an optimal concentration forimmunohistochemical detection of the NPC-1 antigen. A 1:400 dilution ofperoxidase-conjugated streptavidin (Dako North America, Inc.) was thenapplied to the slides for about 30 minutes and slides were washed threetimes. A solution of DAB (Zymed) was applied for about 2-3 minutes thenrinsed with PBS. A solution of hematoxylin was then applied for about 3minutes and rinsed with tap water until clear. The slides weredehydrated with xylene and a coverslip was added using Permount®mounting medium. Additional consecutive slides were stained with humancytokeratin AE1/AE3 (Abcam plc) as a positive control, and human IgG1isotype as a negative control (AXXORA, LLC).

All antibodies were biotinylated prior to use and tested independentlyat various concentrations using human tumor tissues known to react withthe antibodies. Primary antibody (NEO-101) was used at about 10 μg/mL,detected with streptavidin-horseradish peroxidase conjugate, and mountedon slides. A positive staining scale ranging from +1 to +5 was appliedto the staining results, measured by light microscopy. Tissues stainedwith NEO-101 were considered positive (+1 to +5) for an average of 79%of the patient tumor samples (30 of 38 of both colorectal and pancreaticcancer biopsies) including the 5 pancreatic tumor samples. Tissues thatwere negative or considered weak staining by the immunohistochemist wereconsidered negative. These staining results are similar to results fromseveral other studies completed with antibodies using tissue arrayslides, and both frozen and paraffin-embedded surgical specimens.Results of the IHC staining are shown in Table 8.

TABLE 8 IHC Staining Results Number of % Positive by IHC Cancer TypeSubjects with NEO-1O1 Colorectal 33 76% (25/33) Pancreatic 5 100% (5/5)Notes: (1) most tissue biopsy samples were collected when patients werestaged with stage 2 to 4 cancer, (2) negative and positive controltissues slides were included and shown to stain negatively withsecondary antibody only (negative) or anti-cytokeratin antibody(positive).

The IHC staining results using the NEO-101 antibody was then compared tothe results for each serum ELISA for every subject where both sets ofresults (sera and biopsy) were available. For simplicity, the average ofthe serum ELISA from each blood draw was used for this comparison. Theresults of this analysis demonstrated that 84% (32/38) of the serumsamples were positive using a cutoff of 335 units/mL and 79% (30/38) ofthe tissue samples were positive, providing a high concordance of thetwo assays using NEO-101.

A larger number of serum samples were procured to test the utility ofthe serum-based ELISA in detecting the NPC-1 antigen. A sampling of 41colorectal or pancreas cancer patient sera was compared with seracollected from 28 normal healthy blood donors. In this population ofcancer patients, blood was collected serially during an approximately3-month period for several of the patients while they were undergoingvarious treatment regimens with a medical oncologist. For multiplereasons, blood was not collected from all patients at all threetimepoints. Thus, there were 41 patients that donated blood at theirfirst evaluation by the medical oncologist, followed by 33 patients thatdonated their blood at the second visit, and 25 patients who completedall three blood donations at the third visit. The majority of patientswere diagnosed with Stage III or IV disease.

FIG. 2A shows the results of testing this larger panel of colorectal andpancreatic cancer patient serum specimens, compared to a group of normalhealthy blood donors. Analysis of the results demonstrated approximatelya 0.7 log difference between the cancer patients and the healthy donorsat each of the three blood draws. The mean and standard error of themean for each control group for the assays are: Normals (355±60),Col/Pan Ca, 1-month (1,757±580), Col/Pan Ca, 2-month (1,894±671),Col/Pan Ca, 3-month (1,293±390). Using the unpaired t-test (2-tailed)method to evaluate the difference between the Normal sera group and thecancer sera groups, the differences for each comparison were: Normal vs.1-month [p=0.0511]; Normal vs. 2-month [p=0.0397]; Normal vs. 3-month[p=0.0153]. Furthermore, using a cutoff value of 355 cells/well derivedfrom the Normal sera average, 73% of Col/Pan Ca, 1-month sera were abovethe cutoff (30 of 41 samples), and 88% were above the cutoff in each ofthe 2-month (29 of 33 samples) and 3-month (22 of 25 samples) in thosegroups. Overall, the samples represent an average of 82% positive abovethe cutoff established for the assay. These results show that the NPC-1antigen ELISA can distinguish differences between serum from normaldonors and colorectal or pancreas cancer patients, with a good level ofconfidence.

The cancer patient population tested in this study was furtherstratified by disease type. This analysis, in FIG. 2B, shows that therewas no difference distinguished by the mean NEO-101 ELISA results amongthose patients diagnosed with colorectal cancer (n=36) from thosepatients diagnosed with pancreas cancer (n=5). Both groups separatelydemonstrated approximately 0.7 log units higher NPC-1 epitope expressionlevels compared to the group of healthy donors.

NPC-1 epitope may also be used in monitoring colon or pancreas cancerpatients during the course of a treatment regimen, just as the CEA andCA19-9 assays are used currently. That is, as a surrogate marker for atreatment regimen for a cancer patient (is the patient responding ornot). From patients that donated multiple serum samples, the amount ofNPC-1 antigen biomarker detected in the assay was plotted versus thetime of the blood draw. The results showed that some patients appearedto express similar amounts of the NPC-1 antigen during the 2- or 3-monthperiod when blood was drawn (subjects 5, 14, 15, 19, 25, 28, 29),whereas some patients appeared to experience a 1.5× to 5× increase inNPC-1 antigen expression (subjects 1, 2, 7, 33, 39) or a 1.2× to 3×decrease in the NPC-1 antigen expression (subjects 18, 22, 23, 28, 34,36, 40). The significance of these shifts over time are presentlyunclear, but may be related to the tumor burden of the patient at thetime the blood was drawn, which may be directly related to the specifictreatment regimen of individual patients. The results demonstrate trendsfor certain patients that may reflect cancer regression, progression, orstable disease. Once these data are coupled with the disease status inpatients, the correlation is apparent. Additionally, the NEO-101 assayappears to be better than either of the CEA and CA19-9 assays (i.e.,NPC-J is more sensitive). Additionally, neither the CEA nor CA19-9 seratests can be used to diagnose cancer (as does, for example, the prostateserum antigen test). Hence, the present invention provides for thepredictive value of NPC-1 epitope as a new serum biomarker to diagnoseand monitor treatment of colorectal and pancreatic cancer.

Example 6 NPC-1 Epitope is a Glycotope Comprising an AberrantTumor-Specific Glycosylation Pattern

The MUC5AC epitope was mapped and further characterized in order tobetter elucidate the carbohydrate dependence of NEO-101 antibodybinding. CFPAC-1 supernate (pancreatic cancer cell line CFPAC-1supernate) containing NPC-1 epitope was exhaustively digested withthermolysin which resulted in no detectable activity in Western blotwith NEO-101 antibody.

This was a two part experiment where the antigen (pancreatic cancer cellline CFPAC-1 supernate) was digested with the protease thermolysin(Sigma) at an enzyme:substrate ratio of 1:10. CFPAC supernate in 200 mMTRIS buffer, 500 mM NaCl, 25 mM CaCl₂ pH 7.6 overnight at either about37° C. or 65° C. After digestion enzyme inactivated sample (in EDTA)were run in SDS-PAGE gels after which time a conventional western blotwas performed with NEO-101 antibody and anti-human IgG peroxidase(Jackson Laboratories) detection. There was no longer detectable antigenactivity after thermolysin digestion.

The digested CFPAC-1 supernate antigen still retained full inhibitoryactivity in a competition immunoassay where CFPAC-1 supernate antigen iscoated onto a microplate and the binding of soluble NPC-1-C is followedas the readout. Both the CFPAC-1 supernate and its thermolysin digestwere found to inhibit in a similar fashion but the filtrate from a10,000 dalton cutoff spin filter did not. This suggested that theinhibitory fragment(s) are larger than 10,000 daltons but considerablysmaller than the native antigen seen on the gel.

Thermolysin was then used to fragment MUC5AC. The thermolytic fragmentsfrom the three tandem repeat regions of MUC5AC were the selected inorder to construct a multiple alignment of possible epitope containingfragments having a common consensus sequence. These experiments not onlylimited the size of the prospective epitope but also suggested apossible association with O-linked carbohydrate substitution given thepresence of motifs. This data indicates that a NEO-100 antibody binds toa region of MUC5AC comprising the peptide sequence of (SEQ ID NO: 34 or35) which serves as a scaffold for an aberrant, tumor-specificglycosylation pattern. This aberrant, tumor-specific glycosylationpattern (secondary structure) is apparently attached to residues in SEQID NO: 34 or 35 contained in the antigen bound by a NEO-100 antibody(e.g., NEO-101, NEO-102, NEO-103).

The carbohydrate dependence of NEO-101 binding was further confirmed byglycosidase enzyme digestions, chemical modifications, and mimicry. Apanel of glycosidases (Northstar Bioproducts) was used to explore apossible change in the ability of CFPAC-1 supernate antigen to inhibitNEO-101 binding to the same antigen immobilized on microplates(competition assay). The commercial enzyme panel comprised a pluralityof enzymes: (a) o-glycosidase, (b) β1->4 galactosidase, (c) PNGase F,α2->3,6,8,9 specific neuraminidase, and (d) β N acetyl glucosaminidase.Of all enzymes tested neuraminidase (3, 6, 8 selective) stood outproducing significant modifications to the antigen. This was observed inthe competition ELISA using CFPAC coated plates and NEO-101.Surprisingly, of o-glycosidase, (b) β1->4 galactosidase, (c) PNGase F,α2->3,6,8,9 specific neuraminidase, and (d) β N acetyl glucosaminidasetreatment, only the neuraminidase digestion eliminated activity of theantigen. Unexpectedly, it was observed that the antigen activity issensitive to neuraminidase, mild sodium periodate oxidation treatment at4° C. (a method that selectively destroys sensitive vicinal diol bondedhydroxyl groups found in sialic acids) also eliminated the binding ofNEO-101 to the MUC5AC.

The results of the neuraminidase digestion result suggest that sialicacid is comprised in the carbohydrate residues which are attached toamino acids in the primary structure of the antigen that constitute theepitope. A neuraminidase from Macrobdella decora (Calbiochem) which isselective for α2->3 linkages was inactive. Only neuraminidase with broadspectrum (α2->3,6,8) from Arthrobacter ureafaciens showed activity.Since α2->8 linked sialic acid is relatively uncommon except in neuronaltissues, the results highly suggest that the epitope contains sialicacid α2->6 type linkages. The periodate treatment further narrows thebinding to include C8 and C9 hydroxyl groups on sialic acid as possiblecontact points with NEO-101. A competition assay comparing CFPAC-1supernatant treated with α2-3 neuraminidase, α2-3, 6, 8 neuraminidase,and sodium periodate to a CFPAC-1 control was also effected. OnlyCFPAC-1 supernatant treated with α2-3 neuraminidase and sodium periodateshowed a lack of binding of the NEO-101 antibody. Thus, the antigendetected in the serum ELISA bound by the NEO-101 antibody is alsosensitive to sodium periodate and α2-3 neuraminidase but not α2-3, 6, 8neuraminidase.

Serum from a normal healthy individual (Normal Serum) or serum from apatient with a Colon Cancer was treated overnight with severalconcentrations of sodium periodate. The reaction was then stopped byaddition of 50% glycerol. The treated samples were then assayed forNPC-1 epitope content by ELISA using NEO-101 antibody in a homologousformat were NEO-101 was both the capture reagent and detector reagent ina biotinylated form.

A form of mimicry was unexpectedly discovered where bovine submaxillarymucin (BSM) (Sigma) bound very well to NEO-101 in ELISA, and westernblot. This cross-reactive antigen provided a source of material tofurther explore the carbohydrate dependence of NEO-101 binding. The BSMreactivity with NEO-101 antibody was sensitive to both periodate andneuraminidase treatments. The competition assay comparing CFPAC-1supernatant with BSM on the ability to inhibit NEO-101 antibody bindingto CFPAC coated plates.

BSM or CFPAC-1 supernatant was treated with sodium periodate andneutralized essentially as described previously. The treated antigenswere then coated onto a microplate which was subsequently probed with atitration of NEO-101 antibody. The readout after an anti-humanIgG-peroxidase (Jackson) secondary antibody binding step was obtainedwith TMB substrate. This shows that BSM and CFPAC-1 supernate antigenboth have a similar periodate sensitivity with respect to the NPC-1epitope. This result is consistent with mild acid hydrolysis experimentswhich points to a common sialic acid partial glycotope.

The binding of NEO-101 to its antigen(s) is salt sensitive, furthersupporting the finding that the binding may have an ionic dependence,contributed by negatively charged sialic acid residues in the antigen.CFPAC-1 supernate coated plate was a capture and the readout was NEO-101antibody binding in the presence of several concentrations of NaCl.

The NPC-1 monoclonal antibody was compared to the Sialyl Tn monoclonalantibody (Abcam) and antibodies that bind the CA19-9 antigen. With BSMcoated plate as capture and variable amount of NEO-101 added, a constantamount of sialyl Tn antibody was added resulting in no competition ofNEO-101 binding. When sialyl Tn was tested in pre-blocking a BSM plate,no such blocking of NEO-101 binding occurred. 50% of the O-liked glycanson BSM have the following sequence which is defined by antibodiesbinding to Sialyl Tn: NeuAca2→6GalNAcα1→Scr/Thr. Selective neuraminidasedigestion showed that the epitope recognized by the NEO-101 antibodycomprises a NeuAca2-6 linkage. Sialyl Tn antibody blocking experimentsdemonstrated that NEO-101 and Sialyl Tn do not share an epitope as thereis no competition for binding between these antibodies (e.g., Sialyl Tnbinds a different epitope). These results also suggest that the epitoperecognized by NEO-101 is sensitive to removal of α2→6,8 linked sialicacid but not α2→3 linked sialic acid, excluding CA19-9 as the antigen.Further, the epitope is sensitive to mild periodate oxidation therebysuggesting that sialic acid C8, C9 hydroxyl groups may be contact sitesto NEO-101 or mucin. Therefore, the sialyl Tn monoclonal antibody doesnot bind the same epitope as a NEO-100 antibody. Further, a NEO-100antibody does not bind to the CA19-9 antigen.

Accordingly, the NPC-1 epitope is sensitive to mild acid hydrolysis,periodate oxidation, and neuraminidase digestion, all treatments knownto elicit a degradative effect on sialic acid, and suggesting thatsialic acid is a key sugar forming part of the glycotope recognized by aNEO-100 antibody. Further, the linkage of sialic acid to the penultimatesugar of the epitope was suggested to be α2->6 rather than α2->3 byvirtue of the epitope destructive effect only seen with neuraminidasefrom Arthrobacter ureafaciens (broad spectrum neuraminidase) and notneuramidase from Macrobdella décora selective only for α2->3 linkages.Additionally, the NEO-101 antibody binds effectively to bovinesubmaxillary mucin (BSM) and proteolytic digest thereof. This suggeststhat a homologous glycotope exists on BSM and there is diminishedrelevance of the peptide part of the molecule. The NPC-1 epitope is saltsensitive, thereby suggesting the importance of charged residues,possibly due to clustered negatively charged sialic acid residues havingthe appropriate ionic character.

Example 7 Pharmacology and Toxicology Data Proposed Mechanism of Actionof NEO-101

The NEO-101 antibody was tested for antibody-dependent cell cytotoxicity(ADCC) activity against several colorectal and pancreatic tumor celltargets in vitro. The ADCC assay measures the amount of cellcytotoxicity that an antibody facilitates in a defined time period bythe release of radiolabelled cytoplasmic proteins into the culturemedium. The data show that in a standard 4-hour 111-Indium release assaythat NEO-101 facilitated the killing of the colorectal and pancreatictumor cell lines. The specific lytic activity of NEO-101 is demonstratedwith an isotype IgG control as well as cell line controls that do notexpress the MUC5AC antigen (DU145 and SK-mel). See Table 9. The specificlytic activity was titratable with the number of effector cells in theassay.

TABLE 9 ADCC Assay: NEO-101 Antibody Killing Against Tumor Cell LinesEffector: Target % Specific Killing (±SEM) Tumor Cell Line Target CellRatio Isotype control Ab NEO-101 Colo-205 (Colorectal)   50:1   9.8 ±1.9 66.7 ± 0.6   25:1   0.8 ± 1.2 46.4 ± 1.6 12.5:1 −0.5 ± 0.1 32.8 ±2.0 SW620 (Colorectal)   50:1   1.6 ± 0.2 63.7 ± 2.9   25:1   3.5 ± 1.861.0 ± 1.8 12.5:1   0.0 ± 0.3 51.5 ± 0.9 SW1463 (Colorectal)   50:1  0.1 ± 1.1 33.8 ± 1.0   25:1 −1.3 ± 0.2 25.5 ± 0.6 12.5:1 −1.2 ± 0.117.9 ± 1.7 LS174T (Colorectal)   50:1 −1.2 ± 0.1 26.8 ± 2.9   25:1 −0.8± 0.1 18.5 ± 4.1 12.5:1 −1.1 ± 0.0   9.5 ± 0.5 AsPC-1 (Pancreatic)  50:1 −0.8 ± 2.9 44.5 ± 6.8   25:1 −7.0 ± 2.2 36.2 ± 2.6 12.5:1 −1.2 ±0.9 26.5 ± 6.7 CFPAC-1 (Pancreatic)   50:1 −1.2 ± 2.3 26.9 ± 1.6   25:1−2.4 ± 0.1 23.2 ± 2.2 12.5:1 −2.0 ± 0.4 11.1 ± 1.6 PANC-1 (Pancreatic)  50:1 −2.2 ± 0.4 46.8 ± 2.1   25:1 −2.5 ± 0.4 33.2 ± 3.3 12.5:1 −3.9 ±0.3 21.2 ± 0.6 SK-MEL (Melanoma)   50:1   2.7 ± 0.7   4.6 ± 1.1   25:1  1.5 ± 0.3   3.3 ± 1.1 12.5:1   1.6 ± 0.4   2.3 ± 0.6 DU145 (Prostate)  50:1 −0.3 ± 0.2 −0.5 ± 0.3   25:1 −0.7 ± 0.1   0.3 ± 0.8 12.5:1 −0.2 ±0.2 −0.3 ± 0.1

These in vitro results demonstrate that the NEO-101 antibody was capableof directing antibody-dependent cell cytotoxicity in the presence ofnormal human PBMCs.

Anti-Tumor Activity

The NEO-101 antibody was tested for anti-tumor activity using the humanAsPC-1 pancreas tumor xenograft model in nude mice. In this activitymodel, mice were implanted with human AsPC-1 tumor cells and allowed toestablish to approximately 20-50 mm³, measurable with a caliper inapproximately 4-6 days. The treatment regimen included intraperitonealinjection of 200 μg of research-grade NEO-101 or a negative controlhuman IgG (Pierce), followed on the next day with an intraperitonealinjection of IL-2-activated normal human PBMCs (approximately 2×10⁷ permouse per injection). Two cycles of treatment were administered suchthat antibody injections occurred on days 5 and 8, and PBMC injectionsoccurred on days 6 and 9 in this study. Throughout the study, the tumorgrowth was monitored twice weekly by measurement with a caliper. Tumorvolume was calculated using the equation: Volume=(width×length)/2, inunits of cubic millimeters. If a tumor reached approximately 800 mm³, orbecame ulcerated or necrotic, the mouse was humanely sacrificed. Thestudy was terminated on study day 35.

FIG. 3 demonstrates the average tumor growth for each group plottedtogether. Tumor growth inhibition was observed during the antibodytreatment phase of the study, and the difference between the NEO-101treated mice and the control groups was statistically significantbeginning on day 13 and continuing for the remainder of the study(P=0.0072 by one-way ANOVA), as indicated by the asterisk on the graph.

This anti-tumor activity study was repeated in a separate study usingthe same AsPC-1 pancreas tumor model and the 200 μg dose of NEO-101antibody. However, in the second study, four cycles of treatment wereadministered instead of two cycles. The antibody was administered ondays 4, 7, 10, and 13 in this study while the PBMCs were injected ondays 5, 8, 11, and 14. All other parameters were kept the same as theprevious study. The data shown in FIG. 4 demonstrate very similar growthinhibition in response to treatment with NEO-101. Tumor inhibition wasevident during the treatment phase of the study, and the differencebetween the NEO-101 treated mice and the human IgG control mice wasstatistically significant beginning on Day 18 and continuing for theremainder of the study (P=0.0044 by one-way ANOVA; n=8 per group). Thefact that these two independent anti-AsPC-1 activity studies yieldedsuch similar results supports the usefulness of the NEO-101 antibody forthe treatment of pancreatic and colorectal cancer.

Since the LS174T colorectal tumor cell line served as a good target invitro in the ADCC assay, this cell line was used in a xenograft tumormodel. The LS174T cells were implanted subcutaneously in nude mice andthe same treatment regimen was administered to these mice. The datashown in FIG. 5 demonstrate that this is a very aggressive tumor in vivesince the study had to be terminated in less than 3 weeks. Nonetheless,we observed a 2-3-fold reduction in tumor growth in NEO-101 treated micecompared to the 2 control groups of mice following the treatment cycles.The anti-tumor effect upon treatment with NEO-101 was significant on thelast day of measuring tumors with P=0.0145 by one-way ANOVA. However,many of the tumors in the control groups became ulcerated and weregreater than 1000 mm³ and the study was terminated.

Cytokine Response

In a preliminary study designed to evaluate potential cytokine responsesin vivo, normal BALB/c mice were injected intravenously with either 3.5mg/kg or 14 mg/kg (5 mice per group) of research-grade NEO-101. Bloodwas collected on Study Day 3 (⁷² hours post-injection) and Study Day 10.Serum was prepared (including from the pre-bleed of each mouse) andtested for the presence of mouse IL-2, IFN-γ, IL-4, and IL-5 in amultiplex bead assay using the SearchLight Array service offered byAushon BioSystems, Inc.

The data demonstrated that there was a small increase in the serumlevels of IL-2 and IL-5, but no appreciable change in IFN-γ or IL-4 onday 3. There appeared to be no dose-dependency related to the increaseof IL-2 or IFN-γ, and the minor elevation of these 2 cytokines showedevidence of beginning to resolve at the day 10 time point. Thus, in thisstudy a small and apparently transient cytokine response was observedthat might be expected upon injection of a bolus of foreign protein intoa mouse.

Antibody Response

Mouse anti-NEO-101 antibody (MAHA) responses were also measured in thisstudy (CB08-5110). The analysis employed an ELISA based assay to detectNEO-101-specific antibodies in mouse serum. The data demonstrated thatnormal BALB/c mice mounted an antibody response against the NEO-101molecule. However, the antibody responses were highly variable on amouse-to-mouse basis, and the overall responses were moderate,suggesting that the NEO-101 antibody was only mildly immunogenic in micedespite the fact that it is comprised in 67% of human IgG sequences.There were no differences between male and female mouse MAHA responses.

Toxicity

A preliminary non-GLP toxicity study using a research-grade preparationof NEO-101 was also conducted. Normal BALB/c mice were Injected with asingle IV dose of saline, or 3, 10, 30, or 100 mg/kg of NEO-101 (n=3female mice per group). In-life parameters measured included bodyweights and clinical observations. Mice were humanely sacrificed 72hours following the injection and specimens were collected for analysis.Post-mortem parameters included macroscopic examination, blood cellcounts, serum chemistries, and histopathological evaluation of selectedmajor organs and tissues. The results of the preliminary studydemonstrated no significant changes in body weight, blood cell counts,and histopathology of 7 major organs and tissues (liver, spleen, kidney,lung, heart, intestine, pancreas). A mild, but statisticallyinsignificant elevation of serum aspartate transaminase (AST) wasobserved in 2 out of 3 mice that received 100 mg/kg of NEO-101. No othertoxicities were detected in these studies potentially associated withNEO-101, including during histopathological examination of the majororgan systems in these mice.

Pharmacokinetics

To determine whether gender impacted the disposition of NEO-101 in vivo,each treatment group contained four males and four females. Clearance,C_(max) and half-life following a single dose of 10 or 100 mg/kg werecompared by non-parametric Mann-Whitney test. No significantgender-specific differences were observed in clearance or C_(max).However, the serum half-life of NEO-101 was shorter in females than inmales. This finding was only significant at the 100 mg/kg dose level(t½:109.5±14.72^(th) versus 285.4±139.5 h, P=0.029). However, it islikely that this is a spurious observation, arising from highinter-animal variability, as this difference in half-life was notreplicated following multiple doses, regardless of dose level.

The data provides useful guidance for the dosing schedule of possibletherapeutic regimes. Mice injected intravenously with 10 mg/kg ofNEO-101 may be used for comparison to the doses used in therapyregiments for humans, for instance. Overall, the disposition of NEO-101antibody in mice is characterized by low clearance, a limited volume ofdistribution and a long elimination half-life. The mean half-life at 10mg/kg was 129 hours (5.4 days) after a single dose, increasing to 279hours (11.6 days) after four doses, which should allow for adequateexposure when dosed every 2-3 weeks in a clinical trial.

Biodistribution

The biodistribution of the NEO-101 antibody was evaluated intumor-bearing mice using radio-labeled antibody. The NEO-101 antibodywas labeled on surface-exposed tyrosines with 125-Iodine and purifiedvia gel filtration chromatography. Nude mice bearing establishedsubcutaneous human pancreatic tumors (CFPAC-1) or colorectal tumors(LS174T) were injected intravenously with the radioiodinated NEO-101 onday 0. Mice were sacrificed on study day 1, 2, 4, and 6. On necropsydays, mice were exsanguinated and major organs (e.g., lungs, intestine,liver, pancreas, spleen, kidneys, blood) including the subcutaneoustumor were collected.

The data show that radiolabeled NEO-101 localized predominantly in theestablished tumor xenografts that are known to express the MUC5AC targetantigen, and, not in other non-target tissues examined. In thepancreatic CPFAC-1 tumor model, NEO-101 uptake was statistically higherin tumors than in any other tissue type at all timepoints, except whencompared to those in blood in females only on day 6. Interestingly, miceharboring the colorectal LS174T tumor demonstrated NEO-101 uptake thatincreased in both sexes reaching the highest levels on day 6. The uptakewas statistically higher in tumors than in any other tissue typeexamined at any timepoint during the study. These studies support thenotion that NEO-101 can traffic to the tumor site following intravenousadministration of the antibody, where it can bind to it target antigen,accumulate at the tumor site, and elicit an anti-tumor effect.

The biodistribution of NEO-101 in CFPAC-1 pancreatic tumor model wasused to study the concentration of the NEO-101 antibody in tumors overthe course of 6 days. Mice were injected with either 3×10⁶ CFPAC-1 andallowed to grow to 50-100 cm³. Afterward, ¹²⁵I-labeled NEO-101 wasinjected at 400 μg/ml in 200 μl of PBS and the mice sacrificed. Organswere harvested and the amount of ¹²⁵I labeled NEO-101 was counted andnormalized to blood. The data demonstrated localization and accumulationof radiolabeled NEO-101 at the site of the tumor in vivo, whereas noneof the major organ systems (e.g., kidneys, spleen, pancreas, stomach,lungs, liver, intestines) exhibited an enrichment of radiolabeledNEO-101.

Biodistribution of NEO-101 in LS174T colorectal tumor model was studiedusing NEO-101 antibody in tumors over the course of 6 days. Mice wereinjected with 3×10⁶ LS174T cells and allowed to grow to 50-100 cm³.Afterward, ¹²⁵I-labeled NEO-101 was injected at 400 μg/ml in 200 μl ofPBS and the mice sacrificed. Organs were harvested and the amount of¹²⁵I-labeled NEO-101 was counted and normalized to blood. The datademonstrate the time-dependent localization and accumulation ofradiolabeled NEO-101 at the site of the tumor in vivo, whereas none ofthe major organ systems (e.g., kidneys, spleen, pancreas, stomach,lungs, liver, intestines) exhibited an enrichment of radiolabeledNEO-101.

In summary, the results, particularly the in vitro ADCC activity and thein vivo anti-tumor activity support the use of NEO-101 as a therapeuticfor cancer patients who express the tumor target antigen, NPC-1. Tissuestaining with NEO-101 revealed a strong positive correlation to colonand pancreas cancer tissues because little or no cross-reactivity withnormal human pancreas or colon tissue, and no cross-reactivity to othernormal tissues was seen. The pharmacokinetic data demonstrate that theNEO-101 serum half-life in mice is within a similar range compared toother therapeutic immunoglobulins, and supports administration of theantibody every two to three weeks. The bio-distribution studydemonstrated the ability of NEO-101 antibody to traffic to, andaccumulate

in established tumors suggesting that a NEO-100 antibody may be used asa delivery vehicle to delivery agents (e.g., cytotoxic agents or labels)directly to tumors.

Example 8 Detection of NPC-1 Epitope in Fecal Samples

Stools are a rich source of cells derived from the gastrointestinaltract, and cancer antigens may be measured in fecal samples usingstandard techniques, e.g., immunochemistry such as ELISA. Kim, et al.(2003) Annals Clin. & Lab. Sci. 33: 32-38; Tøn, et al. (2000) Clin.Chimica Acta. 292: 41-54. A homologous format ELISA that uses NEO-101antibody as both capture and detection reagent was developed. Apreliminary control experiment with human pancreatic CFPAC-1 tumor cellsupernate (containing the NPC-1 antigen) spiked into a healthy stoolsample showed that stool did not interfere with the ELISA. Next, samplesof stool collected during colonoscopy from colorectal cancer patients(n=4), stool from people with small polyps (n=4), stool from people withmultiple polyps (n=2), stool from people with large polyps (n=3), andstool from healthy adults (n=13) were applied to the ELISA. A solubleextract of stool was prepared by detergent lysis and centrifugation. Thelevel of NEO-101-specific NPC-1 epitope measured in this ELISA wascompared among all groups. Table 10 shows data from two independentexperiments in which some samples were spiked with CFPAC-1 cell linederived from pancreas duct carcinoma:

TABLE 10 Detection of NPC-1 epitope in human fecal extracts by ELISAExperiment 1 Experiment 2 extract extract extract extract Sample 1/101/50 1/10 1/50 1 fecal sample from  285*  187  204 159 healthy donor 2fecal sample from pt  291  204  224 181 with celiac disease 3 fecalsample from pt  855  281  723 231 with polyps 4 fecal sample from pt3629  757 3217 624 with colon cancer (hyperplasia) 5 fecal sample frompt 5137 1043 ND ND with colon cancer sample 1 spiked with 10 μl 1944 461 1354 346 CFPAC-1 supernate sample 2 spiked with 10 μl 2045  438 NDND CFPAC-1 supernate sample 3 spiked with 10 μl 3219  582 ND ND CFPAC-1supernate sample 4 spiked with 10 μl 5926 1373 ND ND CFPAC-1 supernatesample 5 spiked with 10 μl 7692 1694 ND ND CFPAC-1 supernate CFPAC-1supernate 2902 ND 2772 ND HTB 35 supernate  143 ND  82 ND *numbersrepresent NPC-1 epitope-positive cell equivalents/mL ND = not doneHTB-35 = NPC-1 epitope negative control supernate

Results using CFPAC-1 supernate as a surrogate source of NPC-1 antigenshowed that the contents of fecal material did not interfere with theability of the ELISA to measure the NEO-101 antibody reactive NPC-1epitope. When extracts of stool were applied to the ELISA, it wasapparent that healthy people did not express NEO-101 antibody reactiveNPC-1 epitope in their stool. The signal in the assay was similar tobackground levels (average about 723 units). In contrast, people withsmall polyps had higher levels (average about 3,819 units); people withmultiple polyps expressed higher levels (average about 7,369 units);people with large polyps had even higher levels (average about 10,189units); and colon cancer patients had the highest Icvcls of NEO-101reactive antigen (average about 175,983 units), more than about 240times the level of NPC-1 epitope compared with healthy people. ELISAusing NEO-101 antibody (to detect NPC-1 epitope) is a specific anduseful assay for the diagnosis and monitoring of pancreas cancer usingstool samples. Inhibitors of NEO-101 antibody ELISA are not present infecal extracts. The assay is titratable and may be quantitative. Seealso FIG. 1.

This data establishes a correlate level of NEO-101 reactive antigen,measured by a novel stool-based ELISA, with colon cancer diseaseprogression. The level of NEO-101-specific NPC-1 epitope detectedincreased concomitantly with the number and size of polyps observedduring colonoscopy, and reached the highest levels in patients withcolon cancer. Thus, this ELISA test provides for early non-invasivediagnostic screening for colorectal cancer using an anti-NEO-101antibody.

Example 10 NEO-101 Antibody Shows Anti-Tumor Effects In Vitro and InVivo

Introduction: NEO-10 is a chimeric monoclonal antibody which may be usedfor the treatment of pancreatic and colorectal cancers. NEO-101 antibodyappears to recognize a variant form of MUC5AC expressed specifically byhuman pancreatic and colorectal tumor tissues and cell lines.

Methods: The NEO-101 antibody was selected from a panel of hybridomasgenerated from mice immunized with semi-purified membrane-associatedproteins derived from biologically screened, pooled human allogeneiccolon cancer tissues. In vitro assays and in vivo studies were performedto characterize the GMP-grade antibody.

Immunohistochemistry (IHC)

Slides were deparaffinized, rehydrated and antigen retrieval wasperformed. Slides were then stained with 10 μg/ml biotinylated NEO-101antibody and then streptavidin-HRP was applied for color development.Slides were counter stained with H.E., hydrated and fixed. The results

demonstrate NEO-101 binding specific for pancreatic or colorectal tumortissue, but no binding to normal pancreas or colon tissue. See Table 11.The specificity of NEO-101 for pancreatic and colorectal tumor tissuewas further shown by staining lung tumor tissue. While there wassignificant binding to these tissues with a commercial antibody thatrecognizes normal MUC5AC, there was no reactivity of NEO-101 with theselung tumor tissues.

Table 11—Specificity of NEO-101 Antibody

FACs data showing NEO-101 antibody binding to colon cancer andpancreatic cancer cell lines. Cells were washed and suspended in either2 μg/ml NEO-101-FITC or isotype control antibody-FITC for 1 hour, washedand then subjected to FACS analysis. Experiments with all cell lineswere repeated at least three times. The NEO-101 antibody reacts withcolorectal and pancreatic tumor tissues, but does not cross-react withnormal human tissues, except for sporadic, weak binding to certain GItract tissues, which may indicate a pre-malignant state. NEO-101antibody binds to cancer cells as observed by immunofluoresence (IF)staining results using a FITC labeled NEO-101 antibody (2 μg/ml) onpancreatic cancer cell line AsPC-1, colorectal cancer cell line LS174T,but does not bind to the lung cancer cell line A549. DAPI was used tostain the nucleus. The IF showed clear specific staining of thepancreatic and colorectal cells, but not the lung cancer cells. Thestaining pattern of these pancreatic and colorectal tumor cells waspredominantly membrane-associated, consistent with the expressionprofile of MUC5AC. See Table 12.

TABLE 12 NEO-101 Antibody binding to Pancreatic and Colorectal TumorCell Lines Isotype Control NEO-101 Tumor Cell Lines (Percent positive)(Percent positive) LS174 3.85 89.72 Colo-205 2.33 94.67 SW480 3.38 58.98CFPAC 1.79 52.56

The NEO-101 antibody exhibits cell-specific binding and ADCC activityagainst human colorectal and pancreatic tumor cells, but not againstcontrol tumor cell lines. In vivo, the anti-tumor activity of NEO-101antibody was tested using pre-established subcutaneous human tumorxenograft models. Surprisingly, the NEO-101 antibody showed significant,and reproducible, anti-tumor action, including some complete tumorregressions.

The results herein show that NEO-101 antibody may bind specifically topancreatic and colon cancer tissue samples and also to cell lines.NEO-101 antibody may induce antibody dependent cell cytotoxicity incolon and pancreatic cells but not in melanoma and prostate cancer. Invivo studies suggest that NEO-101 antibody inhibits tumor growth inxenograft models of pancreatic and colon cancer. Bio-distributionstudies showed that NEO-101 antibody accumulates in the tumor and not inany major organs. There was mild type I and II cytokine responses andexpected? antibody responses in mice treated with NEO-101. Therefore,the NEO-101 antibody is specific for pancreatic and colon cancer, andinduces ADCC activity in in vitro assays and inhibits tumor growth invivo.

Particularly, the available data relating to the NEO-101 antibodyindicates that it should be safe and efficacious, and that it may haveclinical activity in patients whose tumor expresses the variant MUC5ACepitope. Indeed, this antibody should have broad clinical relevance asapproximately 50-70% of human pancreatic and colon tumor tissues expressan NPC-1 antigen (as shown by positive staining).

Example 11 Biopanning

The biopanning technique harnesses recombinant phage display peptidelibraries, typically based on relatively short recombinant peptidesfused to a phage coat protein. Phage display describes a selectiontechnique in which a library of variants of a peptide or protein isexpressed on the outside of a phage virion, while the genetic materialencoding each variant resides on the inside. See, e.g., Sidhu, et al.(2003) Chembiochem 4(1): 14-25; Ferrer, et al. (1999) J. Pept. Res.54(1): 32-42; BouHamden, et al. (1998) J. Biol. Chem. 273(14):8009-8016; and Whaley, et al. (2000) Nature 405(6787): 665-667.

For example, the PH.D.™-12 phage display peptide library is arecombinatorial library of random dodecapeptides fused to the minor coatprotein (pill) of M13 phage. The displayed 12-mer is expressed at theN-terminus, followed by a short spacer peptide (GGGS), and then thewild-typs pill sequence. The library consists of approximately 2.7×10⁹sequences amplified once to yield approximately 100 copies of eachsequence in 10 μl of phage. The 4B6 Id may be used in competitiveimmunoassays to release NEO-101-bound phage in biopanning (e.g., usingthe Ph.D.™-12 M13 library phage display system) to identify NEO-101epitopes. 4B6 is an anti-idiotypic antibody described in InternationalPatent Application No. PCT/US2011/41503.

More specifically, enrichment of clonal phage by phage tittering usedseveral rounds of biopanning. The first round of biopanning captured amixture of NEO-101-biotin and display phage to a streptavidin-coatedplate, and bound phage was released by 100 μg/ml 4B6 in 0.1% Tween® 20(polysorbate 20 non-ionic detergent), and yielded 1.6×10³ pfu/10 μl.Another round of biopanning, NEO-101 antibody was coupled to Dynal®beads, and bound phage was released by 100 μg/ml 4B6 in 0.1% Tween® 20,yielding 1.1×10⁶ pfu/10 μl. Another round of biopanning captured amixture of NEO-101 antibody and phage to Protein G-agarose, and boundphage was released by 200 μg/ml of 4B6 in 0.5% Tween® 20, which resultedin 4.5×10⁵ pfu/10 μl. Yet another round of biopanning captured a mixtureof NEO-101 antibody and phage to Protein A-agarose, and bound phage wasreleased by 200 μg/ml 4B6 in 0.5% Tween® 20, and yielded 1.3×10⁶ pfu/10μl. Twelve phage clones from each of three biopanning rounds wereselected for sequence analysis. The resulting peptides are shown in FIG.6.

The M13 clones identified from the several rounds of biopanning wereELISA-tested. Aliquots of 100 l/well NEO-101 (1 μg/ml in CBS buffer) in96 well plate were incubated at 4° C. overnight. Wells were then washedthree times with TBST (0.05% Tween). Non-specific binding was inhibitedby blocking with 200 μl of 5% BSA in 0.1 M NaHCO₃ at room temperature(RT) for 1 hr. Clonal M13 was diluted 1:60 in TBS and added at 100μl/well, and the wells incubated at RT for 1 hr. The wells were thenwashed three times with TBST. Aliquots of 100 μl anti-M13-HRP, diluted1:5,000 in TBS, were added to each well, and incubated at RT for 1 hr.The wells were then washed three times, TMB was added, and the plateswere read at 450 nm. Control wells did not contain NEO-101 antibody. Theresults of the clonal M13 binding ELISA are shown in FIG. 7, whichindicated 6 clonal M13 phage bind to NPC-1C coated wells not BSA blockedcontrol wells significantly.

The peptides identified in the biopanning experiments were used incompetitive binding immunoassays using colon cancer antigens. Briefly,the antigen preparation (Colon Ag) was derived from pooled allogeneiccolon cancer specimens from multiple patients, which was obtainedpost-operatively. Cell membranes were isolated from the tumor, andsoluble membrane proteins were prepared by sonication and Sephadex G-200chromatography. Semi-purified tumor-associated antigens were Identifiedby in vitro and in vivo testing in colon cancers and controls forcell-mediated immunoreactivities. Tumor-associated antigens weredetected in fetal intestine and cell membranes, and was localized ontumor cell membranes. Using discontinuous, gradient gel electrophoresis,both tumor-associated antigens and CEA were separated andcross-compared. The tumor-associated antigens (Colon Ag) was shown to bedistinct from CEA (Hollinshead et al., 177 Science 887 (1972).

Aliquots of 100 μl/well Colon Ag (3 μg/ml in CBS buffer) in 96 wellplate were incubated at 4° C. overnight. Wells were then washed threetimes with TBST (0.05% Tween). Non-specific binding was inhibited byblocking with 200 μl of 1% milk in TBS at RT for 1 hr. Aliquots of 100μl/well premixed NEO-101-biotin and test peptide were added, and thewells incubated at RT for 1 hr. The wells were then washed three timeswith TBST. Aliquots of 100 μl streptavidin-HRP, diluted 1:2000, wereadded to each well, and incubated at RT for 1 hr. The wells were thenwashed three times, TMB was added, and the plates were read at 450 nm.Control wells contained NEO-101-biotin at a two-fold serial dilution.The results of the clonal M13 binding ELISA are shown in FIGS. 8A-B.FIG. 8A demonstrated 5/9 clonal M13 phages block NPC-1C binding to colonAg significantly; the percentage of inhibition is more than 60%. The ODvalues from this competitive ELISA test was shown in FIG. 8B.

The M13 clones identified by the biopanning were also immunoassayedusing magnetic beads. Aliquots of 5 μl M13 phage (10¹¹/10 μl) were mixedwith 10 μl NEO-101-coupled Dynal® beads at RT for 20 min on a rotator.CFPAC1 cells (30 μl of 10⁶ cells/ml) were added and incubated at 4° C.for 30 min. Rosetted cells were counted (>8 beads/cell). Results areshown in Table 13. The experiments showed that clones 4-1-2-C5,4-1-3-C8, 4-1-3-C9, 4-1-4-C11 and 4-1-4-C12 blocked NEO-101-coupledbeads from binding to the CFPAC1 cells.

TABLE 13 Binding of M13 clones to NEO-101 antibody-coupled beads. Dynalbeads Positive coupled with: M13 Clone rosette cells (%) H-IgG TBS 1NEO-101 TBS 41 H16C3 TBS 60 NEO-101 4-1-2-C5 0 NEO-101 4-1-3-C8 0NEO-101 4-1-3-C9 0 NEO-101 4-1-3-C11 38 NEO-101 4-1-4-C11 0 NEO-1014-1-4-C12 0 NEO-101 4-1-4-C11 + 4-1-2-C5 0 H16C3 4-1-4-C11 36 H16C34-1-4-C11 + 4-1-2-C5 59

In another experiment, wells were coded with 100 μl/well Colon Ag (3μg/ml in CBS buffer) and incubated at 4° C. overnight, and the wellswashed three times with TBST (0.05% Tween). The wells were blocked with200 μl of 1% milk at RT for 1 hr. A pre-mix of NEO-101-biotin and clonalM13 phages e was added at 100 μl/well and incubated at RT of 1 hr,followed by three washes with TBST. Streptavidin-HRP (100 μl of 1:2000dilution) was added and the reaction incubated at RT for 1 hr, and thewells washed three time. TMB was added, and the wells were read at 450nm. The results of clonal M13 phages blocking NPC-1C binding to colon Agare shown in FIGS. 9A and 9B, which demonstrated the does dependentinhibition of clonal M13 phages on NPC-1C binding to colon Ag inseparate experiments.

Several clones were identified by the biopanning and subsequentimmunoassays. From this work, four clones (and one control) wereselected to synthesizing peprides for further characterization as shownin Table 14. The M13 native sequences are the YSHS (SEQ ID NO: 25) atthe N-terminal end of the peptide, and the GGGS (SEQ ID NO: 26) at theC-terminus of the peptide.

TABLE 14 NEO-1O1-binding peptides identified inbiopanning and immunoassays SEQ Peptide ID Amino Acid Sequence ID NO4-13-C9 YSHSFPEDYFRYTNQKGGGS 19 4-1-4-C12 YSHSSLPDDWFRYINYGGGS 204-1-2-C5 YSHSWHTLPEKSLDENGGGS 21 4-1-3-C8 YSHSWHTLPESGEVTSGGGS 224-1-3-C11 YSHSVHAIEDNWSPRGGGGS 23 (control peptide)

The peptide binding was analyzed in ELISA by adding 100 μl/well ofpeptide in CBS buffer to 96 well plate and incubating at 4° C.overnight, followed by three washes with TBST (0.05% Tween). Thesolution was blocked with 200 μl of 1% milk in TBS at RT for 20 min.Aliquots of 100 μl NEO-101-biotin (1 g/ml) were added, and incubated atRT of 2 hours, followed by three washes with TBST. Streptavidin-HRP (100μl of 1:2000 diluted in 1:10 diluted blocking buffer) was added, and thewells incubated at RT for 1 hr, followed by three washed. TMB was thenadded, and the wells were read at 450 nm. Controls used 4B6-coatedwells. The results of peptide binding to NEO-101 are shown in FIG. 10,which indicates that the peptides failed to bind adequately to the ELISAplate.

To solve the poor binding of peptides to 96 well plate, peptides werebiotinylated using EZ-link micro NHS Biotinylation kit (Pierce Cat#21955), and labeling efficiency checked by dot blot (50%). Thebiotinylated peptide binding was analyzed in ELISA by adding 100 μl/wellof NEO-101 (1 μg/ml) in CBS buffer to 96 well plate and incubating at 4°C. overnight, followed by three washes with TBST (0.05% Tween). Thesolution was blocked with 200μ/of 1% milk in TBS at RT for 20 min.Aliquots of 100 μl peptide-biotin (1 g/ml) in 0.1% milk-TBS were added,and incubated at RT of 2 hr, followed by three washes with TBST.Streptavidin-HRP (100 μl of 1:2000 diluted in 1:10 diluted blockingbuffer) was added, and the wells incubated at RT for 1 hr, followed bythree washed. TMB was then added, and the wells were read at 450 nm.Positive controls for NPC-1C binding used 4B6-biotin. The results areshown in FIG. 11, which indicates biotinylated peptides bind to NPC-1Ccoated plate.

An ELISA analysis of the NEO-101 binding inhibition by peptide 4-1-3-C9and 4-1-4-C12 against Colon Ag was conducted. Aliquots of 100 μl/wellColon Ag (3 μg/ml in CBS buffer) in 96 well plate were incubated at 4°C. overnight, and the wells washed three times with TBST (0.05% Tween).The wells were blocked with 200 μl of 1% milk at RT for 1 hour. Apre-mix of NEO-101-biotin and test peptide was added at 100 μl/well andincubated at RT of 1 hour, followed by three washes with TBST.Streptavidin-HRP (100 μl of 1:2000 dilution) was added and the reactionincubated at RT for 1 hour, and the wells washed three time. TMB wasadded, and the wells were read at 450 nm. The control wasNEO-101-biotin. See FIG. 12. A summary of the data is shown in Table 15:

TABLE 15 Synthetic peptides immunoassay data. Blocking NEO- of NEO-101101 antibody SEQ Peptide bind- binding to ID ID Amino Acid ing colon AgNO 4-1-3- YSHSFPEDYFRYTNQKG Yes Yes 19 C9 GGS 4-1-4- YSHSSLPDDWFRYINYGYes Yes 20 C12 GGS 4-1-3- YSHSWHTLPESGEVTSG Yes No 22 C8 GGS 4-1-3-YSHSVHAIEDNWSPRGG No No 23 C11 GGS (negative control)An additional two peptides were synthesized for confirmation:4-1-4-C12-biotin YSHSSLPDDWFRYINYGGGS-Biotin 20 4-1-4C12-R2SLPDDWFRYINYSLPDDWFRYINY 24 (repeat 12mer)

The peptides thus identified were compared with the MUC5AC sequence, forexample GeneID: 4586 (Homo sapiens), and the results are shown in FIG.13. Among these three peptide, shared amino acids are underlined asfollows:

TABLE 16 Comparison of Three Peptidomimetics Peptide IDAmino Acid Sequence SEQ ID NO 4-1-3-C9 YSHSFPEDYFRYTNQKGGGS 19 4-1-4-C12YSHSSLPDDWFRYINYGGGS 20 4-1-4C12-R2 SLPDDWFRYINYSLPDDWFRYINY 24

On this basis, it was concluded that the following peptidomimetics areantibody-binding peptides that bind NEO-101: SX¹PX²DX³FRYX⁴NX⁵ (SEQ IDNO: 1) wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is T or I andX⁵ is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO: 2) wherein X¹ is for L; X²is E or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y andSLEPEX¹DWX²FRYX³NY (SEQ ID NO: 3) wherein X¹ is E or D; X² is W or Y;and X³ is T or I. Exemplary peptidomimetics include but are not limitedto FPEDYFRYTNQK (SEQ ID NO: 4) and SLPDDWFRYINY (SEQ ID NO: 5) andadditional peptidomimetics are described in SEQ ID NOs: 6-24.

Next, the inhibition of NEO-101 beads binding to CFAC1 Cells (rosettedcells) by peptides 4-1-4C12 and 4-1-4C12-R2 was investigated, as shownin FIG. 14A. FIG. 14B was the results from positive control with 4B6 incompetitive beads assay. As an alternative to observing rosetted cells,the supernatant from CFAC1 cultured cells was used to coat ELISA plates,and a series of competition assays were performed as shown in FIG. 15A-B. Both peptide 4-1-3C9 and 4-1-4C12-R2 showed significant competitionagainst bound CFPAC1 antigen with NEO-101 binding, with peptide 4-1-4C12exhibiting higher inhibition in low concentration comparing with4-1-3C9. Therefore, the peptide 4-1-4-C12 is a peptidomimetic of theNPC-1 epitope and may be used to elicit a NPC-1 specific antibodies.

Example 12 4-1-4-C12 Peptidomimetic Conjugated to KLH is Immunogenic

An exemplary NPC-1 antigen peptidomimetic, 4-1-4C12 (SEQ ID NO: 5) wasconjugated to KLH (Keyhole Limpit Hemocyannin) (“4-1-4-C12-KLH”). Thepeptidomimetic-KLH conjugate, 4-1-4-C12-KLH, binds to NEO-101 antibodyas shown in ELISA assays. Further, 4-1-4C12-KLH blocks NPC-1 binding toNPC-1C antigen specifically in competitive ELISA. After confirming thefunction of 4-1-4C12-KLH, 4-1-4C12-KLH was injected into rabbits. Theimmunized serum after third injection was tested by binding ELISA tocheck the function of 4-1-4C12-KLH induced antibody. The plate wascoated with 4-1-4C12 peptide or CFPAC1 sup, diluted serum was added tothe washed and blocked the plate. Bound rabbit IgG was detected bydonkey anti-rabbit IgG-HRP. 4-1-4C12-KLH immunized serum binds to4-1-4C12 (SEQ ID NO: 5) but also bind to CFPAC1 human pancreatic cellline supernatant in a dose dependent manner. FIG. 16A-B. Thisdemonstrates that a NPC-1 epitope peptidomimetic may be injected into ananimal and elicity an NPC-1 epitope specific immune response.

Purified rabbit IgG (anti 4-1-4C12 pAb) was obtained from immunizedrabbit for confirmation test. The plate was coated with NPC-1C antigen(CFPAC1 or BSM), 4-1-4C12 peptide as control. Anti-4-1-4C12 pAb wasadded to washed and blocked plate. Donkey anti-rabbit IgG-HRP was usedto detect bound rabbit antibody. The results showed that anti 4-1-4C12pAb binds to CFPAC1 human pancreatic cell line supernatant, BSM and4-1-4C12 peptide in a dose dependent manner FIG. 17. Anti 4-1-4C12 pAbhas lower affinity to BSM when comparing with NPC-1C antibody in bindingELISA. FIG. 18.

Therefore, the NPC-1 peptidomimetics described herein may be conjugatedto carriers (e.g., KLH) and retain their antigenicity and elicit animmune response including antibodies which bind the NPC-1 eptiope. Thisimmune response may then be active in clearing (e.g., lysis) NPC-1expressing tumor cells, and thus, have a therapeutic effect in theanimal (e.g., slowing tumor growth, shrinking tumors).

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide comprising a polypeptide at least about 80%identical to the amino acid sequence of SX¹PX²DX³FRYX⁴NX⁵ (SEQ ID NO:1), wherein X¹ is for L; X² is E or D; X³ is Y or W; X⁴ is T or I and X⁵is Q or Y; SX¹PX²DX³FRYX⁴NX⁵K (SEQ ID NO: 2), wherein X¹ is for L; X² isE or D; X³ is Y or W; X⁴ is T or I and X⁵ is Q or Y; SLEPEX¹DWX²FRYX³NY(SEQ ID NO: 3), wherein X¹ is E or D; X² is W or Y; and X³ is T or I;FPEDYFRYTNQK (SEQ ID NO: 4); SLPDDWFRYINY (SEQ ID NO: 5); or any one ofthe amino acid sequences of SEQ ID NOs: 6-24. 2-3. (canceled)
 4. Afusion protein comprising the polypeptide of claim
 1. 5. The fusionprotein of claim 4, wherein said fusion protein comprises a detectablelabel covalently or non-covalently directly or indirectly attachedthereto, wherein optionally said detectable label is selected from thegroup consisting of polyHis tag, FLAG tag, MBP, GST protein, and GFP. 6.(canceled)
 7. A conjugate comprising the polypeptide of claim 1,directly or indirectly, conjugated to a cytotoxic agent, a therapeuticagent, label, carbohydrate, carrier, immunoglobulin or immunoglobulinfragment, or an immunomodulatory agent, wherein optionally saidcarbohydrate is mannose, fucose, glucose, GlcNAs, or maltose; optionallysaid carrier is Keyhole Limpet Hemocyanin (KLH), diphtheria toxoid,cholera toxoid, ovalbumin, bovine serum albumin (BSA), Pseudomonasexoprotein A, or microbial outer membrane proteins (OMPS); optionallysaid label is a chemiluminescent label, paramagnetic label, MRI contrastagent, fluorescent label, bioluminescent label, or radioactive label;optionally said paramagnetic label is aluminum, manganese, platinum,oxygen, lanthanum, lutetium, scandium, yttrium, or gallium; optionallythe cytotoxic agent is a moiety that inhibits DNA, RNA, or proteinsynthesis, a radionuclide, or ribosomal inhibiting protein; oroptionally the cytotoxic agent is ²¹²Bi, ¹³¹I, ¹⁸⁸Re, ⁹⁰Y, vindesine,methotrexate, adriamycin, cisplatin, pokeweed antiviral protein,Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, orcytotoxic phospholipase enzyme. 8-13. (canceled)
 14. A compositioncomprising the polypeptide of claim
 1. 15. The composition of claim 14,further comprising a pharmaceutically acceptable carrier, diagnosticallyacceptable carrier, adjuvant, or excipient. 16-19. (canceled)
 20. Adiagnostic kit comprising the polypeptide of claim
 1. 21-56. (canceled)57. A composition for treating cancer comprising an effective amount ofthe polypeptide of claim
 1. 58-66. (canceled)
 67. A method for treatingcancer comprising administering an effective amount of a polypeptide ofclaim
 1. 68-69. (canceled)
 70. A method for activating dendritic cellscomprising removing dendritic cells from a patient, contacting cells exvivo with an effective amount of a polypeptide of claim 1, andreintroducing activated the dendritic cells into said patient.
 71. Amethod for activating antigen-specific immunity comprising administeringto a patient an effective amount of a polypeptide of claim
 1. 72-86.(canceled)
 87. The method of claim 67, wherein said polypeptide isadministered in combination with another antibody, a lymphokine, or ahematopoietic growth factor.
 88. The method of claim 87, wherein saidagent is administered simultaneously or sequentially with the antibody.89. The method of claim 67, wherein said cancer is lung, breast,ovarian, stomach, pancreas, uterine, esophageal, colorectal, or livercancer.
 90. The method of claim 89, wherein said cancer is a stage 1, 2,3 or 4 cancer.
 91. The method of claim 89, wherein said cancer hasmetastasized.
 92. The method of claim 67, wherein the patient expressesdetectable levels of a NPC-1 epitope.
 93. The method of claim 92,wherein the antigen is detected in a tumor biopsy sample or in theblood, stool, urine, or lymph fluid. 94-131. (canceled)